Corynebacterium glutamicum genes encoding proteins involved in homeostasis and adaptation

ABSTRACT

Isolated nucleic acid molecules, designated HA nucleic acid molecules, which encode novel HA proteins from  Corynebacterium glutamicum  are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing HA nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated HA proteins, mutated HA proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from  C. glutamicum  based on genetic engineering of HA genes in this organism.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/454,437, filed Jun. 4, 2003 which is a continuation of U.S.application Ser. No. 09/602,777, filed Jun. 23, 2000, now U.S. Pat. No.6,831,165, issued Dec. 14, 2004, which claims priority to prior filedU.S. Provisional Patent Application Ser. No. 60/141,031, filed Jun. 25,1999. This application also claims priority to prior filed German PatentApplication No. 19931636.8, filed Jul. 8, 1999, German PatentApplication No. 19932125.6, filed Jul. 9, 1999, German PatentApplication No. 19932126.4, filed Jul. 9, 1999, German PatentApplication No. 19932127.2, filed Jul. 9, 1999, German PatentApplication No. 19932128.0, filed Jul. 9, 1999, German PatentApplication No. 19932129.9, filed Jul. 9, 1999, German PatentApplication No. 19932226.0, filed Jul. 9, 1999, German PatentApplication No. 19932920.6, filed Jul. 14, 1999, German PatentApplication No. 19932922.2, filed Jul. 14, 1999, German PatentApplication No. 19932924.9, filed Jul. 14, 1999, German PatentApplication No. 19932928.1, filed Jul. 14, 1999, German PatentApplication No. 19932930.3, filed Jul. 14, 1999, German PatentApplication No. 19932933.8, filed Jul. 14, 1999, German PatentApplication No. 19932935.4, filed Jul. 14, 1999, German PatentApplication No. 19932973.7, filed Jul. 14, 1999, German PatentApplication No. 19933002.6, filed Jul. 14, 1999, German PatentApplication No. 19933003.4, filed Jul. 14, 1999, German PatentApplication No. 19933005.0, filed Jul. 14, 1999, German PatentApplication No. 19933006.9, filed Jul. 14, 1999, German PatentApplication No. 19941378.9, filed Aug. 31, 1999, German PatentApplication No. 19941379.7, filed Aug. 31, 1999, German PatentApplication No. 19941390.8, filed Aug. 31, 1999, German PatentApplication No. 19941391.6, filed Aug. 31, 1999, and German PatentApplication No. 19942088.2, filed Sep. 3, 1999. The entire contents ofeach of the aforementioned applications are hereby expresslyincorporated herein by this reference.

INCORPORATION OF MATERIAL SUBMITTED ON COMPACT DISCS

This application incorporates herein by reference the material containedon the compact discs submitted herewith as part of this application.Specifically, the file “seqlistcorrected” (1.45 MB) contained on each ofCopy 1, Copy 2 and the CRF copy of the Sequence Listing is herebyincorporated herein by reference. This file was created on Jul. 31,2006. In addition, the files “Appendix A” (430 KB) and “Appendix B” (151KB) contained on each of the compact disks entitled “Appendices Copy 1”and “Appendices Copy 2” are hereby incorporated herein by reference.Each of these files were created on Jul. 31, 2006.

BACKGROUND OF THE INVENTION

Certain products and by-products of naturally-occurring metabolicprocesses in cells have utility in a wide array of industries, includingthe food, feed, cosmetics, and pharmaceutical industries. Thesemolecules, collectively termed ‘fine chemicals’, include organic acids,both proteinogenic and non-proteinogenic amino acids, nucleotides andnucleosides, lipids and fatty acids, diols, carbohydrates, aromaticcompounds, vitamins and cofactors, and enzymes. Their production is mostconveniently performed through the large-scale culture of bacteriadeveloped to produce and secrete large quantities of one or more desiredmolecules. One particularly useful organism for this purpose isCorynebacterium glutamicum, a gram positive, nonpathogenic bacterium.Through strain selection, a number of mutant strains have been developedwhich produce an array of desirable compounds. However, selection ofstrains improved for the production of a particular molecule is atime-consuming and difficult process.

SUMMARY OF THE INVENTION

The invention provides novel bacterial nucleic acid molecules which havea variety of uses. These uses include the identification ofmicroorganisms which can be used to produce fine chemicals, themodulation of fine chemical production in C. glutamicum or relatedbacteria, the typing or identification of C. glutamicum or relatedbacteria, as reference points for mapping the C. glutamicum genome, andas markers for transformation. These novel nucleic acid molecules encodeproteins, referred to herein as homeostasis and adaptation (HA)proteins.

C. glutamicum is a gram positive, aerobic bacterium which is commonlyused in industry for the large-scale production of a variety of finechemicals, and also for the degradation of hydrocarbons (such as inpetroleum spills) and for the oxidation of terpenoids. The HA nucleicacid molecules of the invention, therefore, can be used to identifymicroorganisms which can be used to produce fine chemicals, e.g., byfermentation processes. Modulation of the expression of the HA nucleicacids of the invention, or modification of the sequence of the HAnucleic acid molecules of the invention, can be used to modulate theproduction of one or more fine chemicals from a microorganism (e.g., toimprove the yield or production of one or more fine chemicals from aCorynebacterium or Brevibacterium species).

The HA nucleic acids of the invention may also be used to identify anorganism as being Corynebacterium glutamicum or a close relativethereof, or to identify the presence of C. glutamicum or a relativethereof in a mixed population of microorganisms. The invention providesthe nucleic acid sequences of a number of C. glutamicum genes; byprobing the extracted genomic DNA of a culture of a unique or mixedpopulation of microorganisms under stringent conditions with a probespanning a region of a C. glutamicum gene which is unique to thisorganism, one can ascertain whether this organism is present. AlthoughCorynebacterium glutamicum itself is nonpathogenic, it is related tospecies pathogenic in humans, such as Corynebacterium diphtheriae (thecausative agent of diphtheria); the detection of such organisms is ofsignificant clinical relevance.

The HA nucleic acid molecules of the invention may also serve asreference points for mapping of the C. glutamicum genome, or of genomesof related organisms. Similarly, these molecules, or variants orportions thereof, may serve as markers for genetically engineeredCorynebacterium or Brevibacterium species.

e.g. The HA proteins encoded by the novel nucleic acid molecules of theinvention are capable of, for example, performing a function involved inthe maintenance of homeostasis in C. glutamicum, or in the ability ofthis microorganism to adapt to different environmental conditions. Giventhe availability of cloning vectors for use in Corynebacteriumglutamicum, such as those disclosed in Sinskey et al., U.S. Pat. No.4,649,119, and techniques for genetic manipulation of C. glutamicum andthe related Brevibacterium species (e.g., lactofermentum) (Yoshihama etal, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol.159: 306-311 (1984); and Santamaria et al., J. Gen. Microbiol. 130:2237-2246 (1984)), the nucleic acid molecules of the invention may beutilized in the genetic engineering of this organism to make it a betteror more efficient producer of one or more fine chemicals. This improvedproduction or efficiency of production of a fine chemical may be due toa direct effect of manipulation of a gene of the invention, or it may bedue to an indirect effect of such manipulation.

There are a number of mechanisms by which the alteration of an HAprotein of the invention may directly affect the yield, production,and/or efficiency of production of a fine chemical from a C. glutamicumstrain incorporating such an altered protein. For example, byengineering enzymes which modify or degrade aromatic or aliphaticcompounds such that these enzymes are increased or decreased in activityor number, it may be possible to modulate the production of one or morefine chemicals which are the modification or degradation products ofthese compounds. Similarly, enzymes involved in the metabolism ofinorganic compounds provide key molecules (e.g. phosphorous, sulfur, andnitrogen molecules) for the biosynthesis of such fine chemicals as aminoacids, vitamins, and nucleic acids. By altering the activity or numberof these enzymes in C. glutamicum, it may be possible to increase theconversion of these inorganic compounds (or to use alternate inorganiccompounds) to thus permit improved rates of incorporation of inorganicatoms into these fine chemicals. Genetic engineering of C. glutamicumenzymes involved in general cellular processes may also directly improvefine chemical production, since many of these enzymes directly modifyfine chemicals (e.g., amino acids) or the enzymes which are involved infine chemical synthesis or secretion. Modulation of the activity ornumber of cellular proteases may also have a direct effect on finechemical production, since many proteases may degrade fine chemicals orenzymes involved in fine chemical production or breakdown.

Further, the aforementioned enzymes which participate inaromatic/aliphatic compound modification or degradation, generalbiocatalysis, inorganic compound metabolism or proteolysis are eachthemselves fine chemicals, desirable for their activity in various invitro industrial applications. By altering the number of copies of thegene for one or more of these enzymes in C. glutamicum it may bepossible to increase the number of these proteins produced by the cell,thereby increasing the potential yield or efficiency of production ofthese proteins from large-scale C. glutamicum or related bacterialcultures.

The alteration of an HA protein of the invention may also indirectlyaffect the yield, production, and/or efficiency of production of a finechemical from a C. glutamicum strain incorporating such an alteredprotein. For example, by modulating the activity and/or number of thoseproteins involved in the construction or rearrangement of the cell wall,it may be possible to modify the structure of the cell wall itself suchthat the cell is able to better withstand the mechanical and otherstresses present during large-scale fermentative culture. Also,large-scale growth of C. glutamicum requires significant cell wallproduction. Modulation of the activity or number of cell wallbiosynthetic or degradative enzymes may allow more rapid rates of cellwall biosynthesis, which in turn may permit increased growth rates ofthis microorganism in culture and thereby increase the number of cellsproducing the desired fine chemical.

By modifying the HA enzymes of the invention, one may also indirectlyimpact the yield, production, or efficiency of production of one or morefine chemicals from C. glutamicum. For example, many of the generalenzymes in C. glutamicum may have a significant impact on globalcellular processes (e.g., regulatory processes) which in turn have asignificant effect on fine chemical metabolism. Similarly, proteases,enzymes which modify or degrade possibly toxic aromatic or aliphaticcompounds, and enzymes which promote the metabolism of inorganiccompounds all serve to increase the viability of C. glutamicum. Theproteases aid in the selective removal of misfolded or misregulatedproteins, such as those that might occur under the relatively stressfulenvironmental conditions encountered during large-scale fermentorculture. By altering these proteins, it may be possible to furtherenhance this activity and to improve the viability of C. glutamicum inculture. The aromatic/aliphatic modification or degradation proteins notonly serve to detoxify these waste compounds (which may be encounteredas impurities in culture medium or as waste products from cellsthemselves), but also to permit the cells to utilize alternate carbonsources if the optimal carbon source is limiting in the culture. Byincreasing their number and/or activity, the survival of C. glutamicumcells in culture may be enhanced. The inorganic metabolism proteins ofthe invention supply the cell with inorganic molecules required for allprotein and nucleotide (among others) synthesis, and thus are criticalfor the overall viability of the cell. An increase in the number ofviable cells producing one or more desired fine chemicals in large-scaleculture should result in a concomitant increase in the yield,production, and/or efficiency of production of the fine chemical in theculture.

The invention provides novel nucleic acid molecules which encodeproteins, referred to herein as HA proteins, which are capable of, forexample, performing a function involved in the maintenance ofhomeostasis in C. glutamicum, or of participating in the ability of thismicroorganism to adapt to different environmental conditions. Nucleicacid molecules encoding an HA protein are referred to herein as HAnucleic acid molecules. In a preferred embodiment, an HA proteinparticipates in C. glutamicum cell wall biosynthesis or rearrangements,metabolism of inorganic compounds, modification or degradation ofaromatic or aliphatic compounds, or possesses a C. glutamicum enzymaticor proteolytic activity. Examples of such proteins include those encodedby the genes set forth in Table 1.

Accordingly, one aspect of the invention pertains to isolated nucleicacid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotidesequence encoding an HA protein or biologically active portions thereof,as well as nucleic acid fragments suitable as primers or hybridizationprobes for the detection or amplification of HA-encoding nucleic acids(e.g., DNA or mRNA). In particularly preferred embodiments, the isolatednucleic acid molecule comprises one of the nucleotide sequences setforth in Appendix A or the coding region or a complement thereof of oneof these nucleotide sequences. In other particularly preferredembodiments, the isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes to or is at least about50%, preferably at least about 60%, more preferably at least about 70%,80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%,99% or more homologous to a nucleotide sequence set forth in Appendix A,or a portion thereof. In other preferred embodiments, the isolatednucleic acid molecule encodes one of the amino acid sequences set forthin Appendix B. The preferred HA proteins of the present invention alsopreferably possess at least one of the HA activities described herein.

In another embodiment, the isolated nucleic acid molecule encodes aprotein or portion thereof wherein the protein or portion thereofincludes an amino acid sequence which is sufficiently homologous to anamino acid sequence of Appendix B, e.g., sufficiently homologous to anamino acid sequence of Appendix B such that the protein or portionthereof maintains an HA activity. Preferably, the protein or portionthereof encoded by the nucleic acid molecule maintains the ability toparticipate in the maintenance of homeostasis in C. glutamicum, or toperform a function involved in the adaptation of this microorganism todifferent environmental conditions. In one embodiment, the proteinencoded by the nucleic acid molecule is at least about 50%, preferablyat least about 60%, and more preferably at least about 70%, 80%, or 90%and most preferably at least about 95%, 96%, 97%, 98%, or 99% or morehomologous to an amino acid sequence of Appendix B (e.g., an entireamino acid sequence selected from those sequences set forth in AppendixB). In another preferred embodiment, the protein is a full length C.glutamicum protein which is substantially homologous to an entire aminoacid sequence of Appendix B (encoded by an open reading frame shown inAppendix A).

In another preferred embodiment, the isolated nucleic acid molecule isderived from C. glutamicum and encodes a protein (e.g., an HA fusionprotein) which includes a biologically active domain which is at leastabout 50% or more homologous to one of the amino acid sequences ofAppendix B and is able to participate in the repair or recombination ofDNA, in the transposition of genetic material, in gene expression (i.e.,the processes of transcription or translation), in protein folding, orin protein secretion in Corynebacterium glutamicum, or has one or moreof the activities set forth in Table 1, and which also includesheterologous nucleic acid sequences encoding a heterologous polypeptideor regulatory regions.

In another embodiment, the isolated nucleic acid molecule is at least 15nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule comprising a nucleotide sequence of Appendix A.Preferably, the isolated nucleic acid molecule corresponds to anaturally-occurring nucleic acid molecule. More preferably, the isolatednucleic acid encodes a naturally-occurring C. glutamicum HA protein, ora biologically active portion thereof.

Another aspect of the invention pertains to vectors, e.g., recombinantexpression vectors, containing the nucleic acid molecules of theinvention, and host cells into which such vectors have been introduced.In one embodiment, such a host cell is used to produce an HA protein byculturing the host cell in a suitable medium. The HA protein can be thenisolated from the medium or the host cell.

Yet another aspect of the invention pertains to a genetically alteredmicroorganism in which an HA gene has been introduced or altered. In oneembodiment, the genome of the microorganism has been altered byintroduction of a nucleic acid molecule of the invention encodingwild-type or mutated HA sequence as a transgene. In another embodiment,an endogenous HA gene within the genome of the microorganism has beenaltered, e.g., functionally disrupted, by homologous recombination withan altered HA gene. In another embodiment, an endogenous or introducedHA gene in a microorganism has been altered by one or more pointmutations, deletions, or inversions, but still encodes a functional HAprotein. In still another embodiment, one or more of the regulatoryregions (e.g., a promoter, repressor, or inducer) of an HA gene in amicroorganism has been altered (e.g., by deletion, truncation,inversion, or point mutation) such that the expression of the HA gene ismodulated. In a preferred embodiment, the microorganism belongs to thegenus Corynebacterium or Brevibacterium, with Corynebacterium glutamicumbeing particularly preferred. In a preferred embodiment, themicroorganism is also utilized for the production of a desired compound,such as an amino acid, with lysine being particularly preferred.

In another aspect, the invention provides a method of identifying thepresence or activity of Cornyebacterium diphtheriae in a subject. Thismethod includes detection of one or more of the nucleic acid or aminoacid sequences of the invention (e.g., the sequences set forth inAppendix A or Appendix B) in a subject, thereby detecting the presenceor activity of Corynebacterium diphtheriae in the subject.

Still another aspect of the invention pertains to an isolated HA proteinor a portion, e.g., a biologically active portion, thereof. In apreferred embodiment, the isolated HA protein or portion thereof canparticipate in the maintenance of homeostasis in C. glutamicum, or canperform a function involved in the adaptation of this microorganism todifferent environmental conditions. In another preferred embodiment, theisolated HA protein or portion thereof is sufficiently homologous to anamino acid sequence of Appendix B such that the protein or portionthereof maintains the ability to participate in the maintenance ofhomeostasis in C. glutamicum, or to perform a function involved in theadaptation of this microorganism to different environmental conditions.

The invention also provides an isolated preparation of an HA protein. Inpreferred embodiments, the HA protein comprises an amino acid sequenceof Appendix B. In another preferred embodiment, the invention pertainsto an isolated full length protein which is substantially homologous toan entire amino acid sequence of Appendix B (encoded by an open readingframe set forth in Appendix A). In yet another embodiment, the proteinis at least about 50%, preferably at least about 60%, and morepreferably at least about 70%, 80%, or 90%, and most preferably at leastabout 95%, 96%, 97%, 98%, or 99% or more homologous to an entire aminoacid sequence of Appendix B. In other embodiments, the isolated HAprotein comprises an amino acid sequence which is at least about 50% ormore homologous to one of the amino acid sequences of Appendix B and isable to participate in the maintenance of homeostasis in C. glutamicum,or to perform a function involved in the adaptation of thismicroorganism to different environmental conditions, or has one or moreof the activities set forth in Table 1.

Alternatively, the isolated HA protein can comprise an amino acidsequence which is encoded by a nucleotide sequence which hybridizes,e.g., hybridizes under stringent conditions, or is at least about 50%,preferably at least about 60%, more preferably at least about 70%, 80%,or 90%, and even more preferably at least about 95%, 96%, 97%, 98,%, or99% or more homologous, to a nucleotide sequence of Appendix B. It isalso preferred that the preferred forms of HA proteins also have one ormore of the HA bioactivities described herein.

The HA polypeptide, or a biologically active portion thereof, can beoperatively linked to a non-HA polypeptide to form a fusion protein. Inpreferred embodiments, this fusion protein has an activity which differsfrom that of the HA protein alone. In other preferred embodiments, thisfusion protein participates in the maintenance of homeostasis in C.glutamicum, or performs a function involved in the adaptation of thismicroorganism to different environmental conditions. In particularlypreferred embodiments, integration of this fusion protein into a hostcell modulates production of a desired compound from the cell.

In another aspect, the invention provides methods for screeningmolecules which modulate the activity of an HA protein, either byinteracting with the protein itself or a substrate or binding partner ofthe HA protein, or by modulating the transcription or translation of anHA nucleic acid molecule of the invention.

Another aspect of the invention pertains to a method for producing afine chemical. This method involves the culturing of a cell containing avector directing the expression of an HA nucleic acid molecule of theinvention, such that a fine chemical is produced. In a preferredembodiment, this method further includes the step of obtaining a cellcontaining such a vector, in which a cell is transfected with a vectordirecting the expression of an HA nucleic acid. In another preferredembodiment, this method further includes the step of recovering the finechemical from the culture. In a particularly preferred embodiment, thecell is from the genus Corynebacterium or Brevibacterium, or is selectedfrom those strains set forth in Table 3.

Another aspect of the invention pertains to methods for modulatingproduction of a molecule from a microorganism. Such methods includecontacting the cell with an agent which modulates HA protein activity orHA nucleic acid expression such that a cell associated activity isaltered relative to this same activity in the absence of the agent. In apreferred embodiment, the cell is modulated for one or more C.glutamicum processes involved in cell wall biosynthesis orrearrangements, metabolism of inorganic compounds, modification ordegradation of aromatic or aliphatic compounds, or enzymatic orproteolytic activities. The agent which modulates HA protein activitycan be an agent which stimulates HA protein activity or HA nucleic acidexpression. Examples of agents which stimulate HA protein activity or HAnucleic acid expression include small molecules, active HA proteins, andnucleic acids encoding HA proteins that have been introduced into thecell. Examples of agents which inhibit HA activity or expression includesmall molecules and antisense HA nucleic acid molecules.

Another aspect of the invention pertains to methods for modulatingyields of a desired compound from a cell, involving the introduction ofa wild-type or mutant HA gene into a cell, either maintained on aseparate plasmid or integrated into the genome of the host cell. Ifintegrated into the genome, such integration can be random, or it cantake place by homologous recombination such that the native gene isreplaced by the introduced copy, causing the production of the desiredcompound from the cell to be modulated. In a preferred embodiment, saidyields are increased. In another preferred embodiment, said chemical isa fine chemical. In a particularly preferred embodiment, said finechemical is an amino acid. In especially preferred embodiments, saidamino acid is L-lysine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides HA nucleic acid and protein moleculeswhich are involved in C. glutamicum cell wall biosynthesis orrearrangements, metabolism of inorganic compounds, modification ordegradation of aromatic or aliphatic compounds, or that have a C.glutamicum enzymatic or proteolytic activity. The molecules of theinvention may be utilized in the modulation of production of finechemicals from microorganisms, such as C. glutamicum, either directly(e.g., where overexpression or optimization of activity of a proteininvolved in the production of a fine chemical (e.g., an enzyme) has adirect impact on the yield, production, and/or efficiency of productionof a fine chemical from the modified C. glutamicum), or an indirectimpact which nonetheless results in an increase of yield, production,and/or efficiency of production of the desired compound (e.g., wheremodulation of the activity or number of copies of a C. glutamicumaromatic or aliphatic modification or degradation protein results in anincrease in the viability of C. glutamicum cells, which in turn permitsincreased production in a large-scale culture setting). Aspects of theinvention are further explicated below.

I. Fine Chemicals

The term ‘fine chemical’ is art-recognized and includes moleculesproduced by an organism which have applications in various industries,such as, but not limited to, the pharmaceutical, agriculture, andcosmetics industries. Such compounds include organic acids, such astartaric acid, itaconic acid, and diaminopimelic acid, bothproteinogenic and non-proteinogenic amino acids, purine and pyrimidinebases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A.(1996) Nucleotides and related compounds, p. 561-612, in Biotechnologyvol. 6, Rehm et al., eds. VCH: Weinheim, and references containedtherein), lipids, both saturated and unsaturated fatty acids (e.g.,arachidonic acid), diols (e.g., propane diol, and butane diol),carbohydrates (e.g., hyaluronic acid and trehalose), aromatic compounds(e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors(as described in Ullmann's Encyclopedia of Industrial Chemistry, vol.A27, “Vitamins”, p. 443-613 (1996) VCH: Weinheim and references therein;and Ong, A. S., Niki, E. & Packer, L. (1995) “Nutrition, Lipids, Health,and Disease” Proceedings of the UNESCO/Confederation of Scientific andTechnological Associations in Malaysia, and the Society for Free RadicalResearch—Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press,(1995)), enzymes, polyketides (Cane et al. (1998) Science 282: 63-68),and all other chemicals described in Gutcho (1983) Chemicals byFermentation, Noyes Data Corporation, ISBN: 0818805086 and referencestherein. The metabolism and uses of certain of these fine chemicals arefurther explicated below.

A. Amino Acid Metabolism and Uses

Amino acids comprise the basic structural units of all proteins, and assuch are essential for normal cellular functioning in all organisms. Theterm “amino acid” is art-recognized. The proteinogenic amino acids, ofwhich there are 20 species, serve as structural units for proteins, inwhich they are linked by peptide bonds, while the nonproteinogenic aminoacids (hundreds of which are known) are not normally found in proteins(see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97VCH: Weinheim (1985)). Amino acids may be in the D- or L-opticalconfiguration, though L-amino acids are generally the only type found innaturally-occurring proteins. Biosynthetic and degradative pathways ofeach of the 20 proteinogenic amino acids have been well characterized inboth prokaryotic and eukaryotic cells (see, for example, Stryer, L.Biochemistry, 3^(rd) edition, pages 578-590 (1988)). The ‘essential’amino acids (histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan, and valine), so named because theyare generally a nutritional requirement due to the complexity of theirbiosyntheses, are readily converted by simple biosynthetic pathways tothe remaining 11 ‘nonessential’ amino acids (alanine, arginine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline,serine, and tyrosine). Higher animals do retain the ability tosynthesize some of these amino acids, but the essential amino acids mustbe supplied from the diet in order for normal protein synthesis tooccur.

Aside from their function in protein biosynthesis, these amino acids areinteresting chemicals in their own right, and many have been found tohave various applications in the food, feed, chemical, cosmetics,agriculture, and pharmaceutical industries. Lysine is an important aminoacid in the nutrition not only of humans, but also of monogastricanimals such as poultry and swine. Glutamate is most commonly used as aflavor additive (mono-sodium glutamate, MSG) and is widely usedthroughout the food industry, as are aspartate, phenylalanine, glycine,and cysteine. Glycine, L-methionine and tryptophan are all utilized inthe pharmaceutical industry. Glutamine, valine, leucine, isoleucine,histidine, arginine, proline, serine and alanine are of use in both thepharmaceutical and cosmetics industries. Threonine, tryptophan, andD/L-methionine are common feed additives. (Leuchtenberger, W. (1996)Amino aids—technical production and use, p. 466-502 in Rehm et al.(eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally,these amino acids have been found to be useful as precursors for thesynthesis of synthetic amino acids and proteins, such asN-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan,and others described in Ulmann's Encyclopedia of Industrial Chemistry,vol. A2, p. 57-97, VCH: Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms capable ofproducing them, such as bacteria, has been well characterized (forreview of bacterial amino acid biosynthesis and regulation thereof, seeUmbarger, H. E. (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate issynthesized by the reductive amination of α-ketoglutarate, anintermediate in the citric acid cycle. Glutamine, proline, and arginineare each subsequently produced from glutamate. The biosynthesis ofserine is a three-step process beginning with 3-phosphoglycerate (anintermediate in glycolysis), and resulting in this amino acid afteroxidation, transamination, and hydrolysis steps. Both cysteine andglycine are produced from serine; the former by the condensation ofhomocysteine with serine, and the latter by the transferal of theside-chain β-carbon atom to tetrahydrofolate, in a reaction catalyzed byserine transhydroxymethylase. Phenylalanine, and tyrosine aresynthesized from the glycolytic and pentose phosphate pathway precursorserythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosyntheticpathway that differ only at the final two steps after synthesis ofprephenate. Tryptophan is also produced from these two initialmolecules, but its synthesis is an 11-step pathway. Tyrosine may also besynthesized from phenylalanine, in a reaction catalyzed by phenylalaninehydroxylase. Alanine, valine, and leucine are all biosynthetic productsof pyruvate, the final product of glycolysis. Aspartate is formed fromoxaloacetate, an intermediate of the citric acid cycle. Asparagine,methionine, threonine, and lysine are each produced by the conversion ofaspartate. Isoleucine is formed from threonine. A complex 9-step pathwayresults in the production of histidine from5-phosphoribosyl-1-pyrophosphate, an activated sugar.

Amino acids in excess of the protein synthesis needs of the cell cannotbe stored, and are instead degraded to provide intermediates for themajor metabolic pathways of the cell (for review see Stryer, L.Biochemistry 3^(rd) ed. Ch. 21 “Amino Acid Degradation and the UreaCycle” p. 495-516 (1988)). Although the cell is able to convert unwantedamino acids into useful metabolic intermediates, amino acid productionis costly in terms of energy, precursor molecules, and the enzymesnecessary to synthesize them. Thus it is not surprising that amino acidbiosynthesis is regulated by feedback inhibition, in which the presenceof a particular amino acid serves to slow or entirely stop its ownproduction (for overview of feedback mechanisms in amino acidbiosynthetic pathways, see Stryer, L. Biochemistry, 3^(rd) ed. Ch. 24:“Biosynthesis of Amino Acids and Heme” p. 575-600 (1988)). Thus, theoutput of any particular amino acid is limited by the amount of thatamino acid present in the cell.

B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses

Vitamins, cofactors, and nutraceuticals comprise another group ofmolecules which the higher animals have lost the ability to synthesizeand so must ingest, although they are readily synthesized by otherorganisms such as bacteria. These molecules are either bioactivesubstances themselves, or are precursors of biologically activesubstances which may serve as electron carriers or intermediates in avariety of metabolic pathways. Aside from their nutritive value, thesecompounds also have significant industrial value as coloring agents,antioxidants, and catalysts or other processing aids. (For an overviewof the structure, activity, and industrial applications of thesecompounds, see, for example, Ullman's Encyclopedia of IndustrialChemistry, “Vitamins” vol. A27, p. 443-613, VCH: Weinheim, 1996.) Theterm “vitamin” is art-recognized, and includes nutrients which arerequired by an organism for normal functioning, but which that organismcannot synthesize by itself. The group of vitamins may encompasscofactors and nutraceutical compounds. The language “cofactor” includesnonproteinaceous compounds required for a normal enzymatic activity tooccur. Such compounds may be organic or inorganic; the cofactormolecules of the invention are preferably organic. The term“nutraceutical” includes dietary supplements having health benefits inplants and animals, particularly humans. Examples of such molecules arevitamins, antioxidants, and also certain lipids (e.g., polyunsaturatedfatty acids).

The biosynthesis of these molecules in organisms capable of producingthem, such as bacteria, has been largely characterized (Ullman'sEncyclopedia of Industrial Chemistry, “Vitamins” vol. A27, p. 443-613,VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley & Sons; Ong, A. S., Niki,E. & Packer, L. (1995) “Nutrition, Lipids, Health, and Disease”Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia, and the Society for Free RadicalResearch—Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press:Champaign, Ill. X, 374 S).

Thiamin (vitamin B₁) is produced by the chemical coupling of pyrimidineand thiazole moieties. Riboflavin (vitamin B₂) is synthesized fromguanosine-5′-triphosphate (GTP) and ribose-5′-phosphate. Riboflavin, inturn, is utilized for the synthesis of flavin mononucleotide (FMN) andflavin adenine dinucleotide (FAD). The family of compounds collectivelytermed ‘vitamin B₆’ (e.g., pyridoxine, pyridoxamine,pyridoxa-5′-phosphate, and the commercially used pyridoxinhydrochloride) are all derivatives of the common structural unit,5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid,(R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-β-alanine) can beproduced either by chemical synthesis or by fermentation. The finalsteps in pantothenate) biosynthesis consist of the ATP-drivencondensation of β-alanine and pantoic acid. The enzymes responsible forthe biosynthesis steps for the conversion to pantoic acid, to β-alanineand for the condensation to panthotenic acid are known. Themetabolically active form of pantothenate is Coenzyme A, for which thebiosynthesis proceeds in 5 enzymatic steps. Pantothenate,pyridoxal-5′-phosphate, cysteine and ATP are the precursors of CoenzymeA. These enzymes not only catalyze the formation of panthothante, butalso the production of (R)-pantoic acid, (R)-pantolacton, (R)-panthenol(provitamin B₅), pantetheine (and its derivatives) and coenzyme A.

Biotin biosynthesis from the precursor molecule pimeloyl-CoA inmicroorganisms has been studied in detail and several of the genesinvolved have been identified. Many of the corresponding proteins havebeen found to also be involved in Fe-cluster synthesis and are membersof the nifS class of proteins. Lipoic acid is derived from octanoicacid, and serves as a coenzyme in energy metabolism, where it becomespart of the pyruvate dehydrogenase complex and the α-ketoglutaratedehydrogenase complex. The folates are a group of substances which areall derivatives of folic acid, which is turn is derived from L-glutamicacid, p-amino-benzoic acid and 6-methylpterin. The biosynthesis of folicacid and its derivatives, starting from the metabolism intermediatesguanosine-5′-triphosphate (GTP), L-glutamic acid and p-amino-benzoicacid has been studied in detail in certain microorganisms.

Corrinoids (such as the cobalamines and particularly vitamin B₁₂) andporphyrines belong to a group of chemicals characterized by atetrapyrole ring system. The biosynthesis of vitamin B₁₂ is sufficientlycomplex that it has not yet been completely characterized, but many ofthe enzymes and substrates involved are now known. Nicotinic acid(nicotinate), and nicotinamide are pyridine derivatives which are alsotermed ‘niacin’. Niacin is the precursor of the important coenzymes NAD(nicotinamide adenine dinucleotide) and NADP (nicotinamide adeninedinucleotide phosphate) and their reduced forms.

The large-scale production of these compounds has largely relied oncell-free chemical syntheses, though some of these chemicals have alsobeen produced by large-scale culture of microorganisms, such asriboflavin, Vitamin B₆, pantothenate, and biotin. Only Vitamin B₁₂ isproduced solely by fermentation, due to the complexity of its synthesis.In vitro methodologies require significant inputs of materials and time,often at great cost.

C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Purine and pyrimidine metabolism genes and their corresponding proteinsare important targets for the therapy of tumor diseases and viralinfections. The language “purine” or “pyrimidine” includes thenitrogenous bases which are constituents of nucleic acids, co-enzymes,and nucleotides. The term “nucleotide” includes the basic structuralunits of nucleic acid molecules, which are comprised of a nitrogenousbase, a pentose sugar (in the case of RNA, the sugar is ribose; in thecase of DNA, the sugar is D-deoxyribose), and phosphoric acid. Thelanguage “nucleoside” includes molecules which serve as precursors tonucleotides, but which are lacking the phosphoric acid moiety thatnucleotides possess. By inhibiting the biosynthesis of these molecules,or their mobilization to form nucleic acid molecules, it is possible toinhibit RNA and DNA synthesis; by inhibiting this activity in a fashiontargeted to cancerous cells, the ability of tumor cells to divide andreplicate may be inhibited. Additionally, there are nucleotides which donot form nucleic acid molecules, but rather serve as energy stores(i.e., AMP) or as coenzymes (i.e., FAD and NAD).

Several publications have described the use of these chemicals for thesemedical indications, by influencing purine and/or pyrimidine metabolism(e.g. Christopherson, R. I. and Lyons, S. D. (1990) “Potent inhibitorsof de novo pyrimidine and purine biosynthesis as chemotherapeuticagents.” Med. Res. Reviews 10: 505-548). Studies of enzymes involved inpurine and pyrimidine metabolism have been focused on the development ofnew drugs which can be used, for example, as immunosuppressants oranti-proliferants (Smith, J. L., (1995) “Enzymes in nucleotidesynthesis.” Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem Soc.Transact. 23: 877-902). However, purine and pyrimidine bases,nucleosides and nucleotides have other utilities: as intermediates inthe biosynthesis of several fine chemicals (e.g., thiamine,S-adenosyl-methionine, folates, or riboflavin), as energy carriers forthe cell (e.g., ATP or GTP), and for chemicals themselves, commonly usedas flavor enhancers (e.g., IMP or GMP) or for several medicinalapplications (see, for example, Kuninaka, A. (1996) Nucleotides andRelated Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH:Weinheim, p. 561-612). Also, enzymes involved in purine, pyrimidine,nucleoside, or nucleotide metabolism are increasingly serving as targetsagainst which chemicals for crop protection, including fungicides,herbicides and insecticides, are developed.

The metabolism of these compounds in bacteria has been characterized(for reviews see, for example, Zalkin, H. and Dixon, J. E. (1992) “denovo purine nucleotide biosynthesis”, in: Progress in Nucleic AcidResearch and Molecular Biology, vol. 42, Academic Press: p. 259-287; andMichal, G. (1999) “Nucleotides and Nucleosides”, Chapter 8 in:Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,Wiley: New York). Purine metabolism has been the subject of intensiveresearch, and is essential to the normal functioning of the cell.Impaired purine metabolism in higher animals can cause severe disease,such as gout. Purine nucleotides are synthesized fromribose-5-phosphate, in a series of steps through the intermediatecompound inosine-5′-phosphate (IMP), resulting in the production ofguanosine-5′-monophosphate (GMP) or adenosine-5′-monophosphate (AMP),from which the triphosphate forms utilized as nucleotides are readilyformed. These compounds are also utilized as energy stores, so theirdegradation provides energy for many different biochemical processes inthe cell. Pyrimidine biosynthesis proceeds by the formation ofuridine-5′-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, isconverted to cytidine-5′-triphosphate (CTP). The deoxy-forms of all ofthese nucleotides are produced in a one step reduction reaction from thediphosphate ribose form of the nucleotide to the diphosphate deoxyriboseform of the nucleotide. Upon phosphorylation, these molecules are ableto participate in DNA synthesis.

D. Trehalose Metabolism and Uses

Trehalose consists of two glucose molecules, bound in α,α-1,1 linkage.It is commonly used in the food industry as a sweetener, an additive fordried or frozen foods, and in beverages. However, it also hasapplications in the pharmaceutical, cosmetics and biotechnologyindustries (see, for example, Nishimoto et al., (1998) U.S. Pat. No.5,759,610; Singer, M. A. and Lindquist, S. (1998) Trends Biotech. 16:460-467; Paiva, C. L. A. and Panek, A. D. (1996) Biotech. Ann. Rev. 2:293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose isproduced by enzymes from many microorganisms and is naturally releasedinto the surrounding medium, from which it can be collected usingmethods known in the art.

II. Maintenance of Homeostasis in C. glutamicum and EnvironmentalAdaptation

The metabolic and other biochemical processes by which cells functionare sensitive to environmental conditions such as temperature, pressure,solute concentration, and availability of oxygen. When one or more suchenvironmental condition is perturbed or altered in a fashion that isincompatible with the normal functioning of these cellular processes,the cell must act to maintain an intracellular environment which willpermit them to occur despite the hostile extracellular environment. Grampositive bacterial cells, such as C. glutamicum cells, have a number ofmechanisms by which internal homeostasis may be maintained despiteunfavorable extracellular conditions. These include a cell wall,proteins which are able to degrade possibly toxic aromatic and aliphaticcompounds, mechanisms of proteolysis whereby misfolded or misregulatedproteins may be rapidly destroyed, and catalysts which permitintracellular reactions to occur which would not normally take placeunder the conditions optimal for bacterial growth.

Aside from merely surviving in a hostile environment, bacterial cells(e.g. C. glutamicum cells) are also frequently able to adapt such thatthey are able to take advantage of such conditions. For example, cellsin an environment lacking desired carbon sources may be able to adapt togrowth on a less-suitable carbon source. Also, cells may be able toutilize less desirable inorganic compounds when the commonly utilizedones are unavailable. C. glutamicum cells possess a number of geneswhich permit them to adapt to utilize inorganic and organic moleculeswhich they would normally not encounter under optimal growth conditionsas nutrients and precursors for metabolism. Aspects of cellularprocesses involved in homeostasis and adaptation are further explicatedbelow.

A. Modification and Degradation of Aromatic and Aliphatic Compounds

Bacterial cells are routinely exposed to a variety of aromatic andaliphatic compounds in nature. Aromatic compounds are organic moleculeshaving a cyclic ring structure, while aliphatic compounds are organicmolecules having open chain structures rather than ring structures. Suchcompounds may arise as by-products of industrial processes (e.g.,benzene or toluene), but may also be produced by certain microorganisms(e.g., alcohols). Many of these compounds are toxic to cells,particularly the aromatic compounds, which are highly reactive due tothe high-energy ring structure. Thus, certain bacteria have developedmechanisms by which they are able to modify or degrade these compoundssuch that they are no longer hazardous to the cell. Cells may possessenzymes that are able to, for example, hydroxylate, isomerize, ormethylate aromatic or aliphatic compounds such that they are eitherrendered less toxic, or such that the modified form is able to beprocessed by standard cellular waste and degradation pathways. Also,cells may possess enzymes which are able to specifically degrade one ormore such potentially hazardous substance, thereby protecting the cell.Principles and examples of these types of modification and degradationprocesses in bacteria are described in several publications, e.g., Sahm,H. (1999) “Procaryotes in Industrial Production” in Lengeler, J. W. etal., eds. Biology of the Procaryotes, Thieme Verlag: Stuttgart; andSchlegel, H. G. (1992) Allgemeine Mikrobiologie, Thieme: Stuttgart).

Aside from simply inactivating hazardous aromatic or aliphaticcompounds, many bacteria have evolved to be able to utilize thesecompounds as carbon sources for continued metabolism when the preferredcarbon sources of the cell are not available. For example, Pseudomonasstrains able to utilize toluene, benzene, and 1,10-dichlorodecane ascarbon sources are known (Chang, B. V. et al. (1997) Chemosphere 35(12):2807-2815; Wischnak, C. et al. (1998) Appl. Environ. Microbiol. 64(9):3507-3511; Churchill, S. A. et al. (1999) Appl. Environ. Microbiol.65(2): 549-552). There are similar examples from many other bacterialspecies which are known in the art.

The ability of certain bacteria to modify or degrade aromatic andaliphatic compounds has begun to be exploited. Petroleum is a complexmixture of chemicals which includes aliphatic molecules and aromaticcompounds. By applying bacteria having the ability to degrade or modifythese toxic compounds to an oil spill, for example, it is possible toeliminate much of the environmental damage with high efficiency and lowcost (see, for example, Smith, M. R. (1990) “The biodegradation ofaromatic hydrocarbons by bacteria” Biodegradation 1(2-3): 191-206; andSuyama, T. et al. (1998) “Bacterial isolates degrading aliphaticpolycarbonates,” FEMS Microbiol. Lett. 161(2): 255-261).

B. Metabolism of Inorganic Compounds

Cells (e.g., bacterial cells) contain large quantities of differentmolecules, such as water, inorganic ions, and organic substances (e.g.,proteins, sugars, and other macromolecules). The bulk of the mass of atypical cell consists of only 4 types of atoms: carbon, oxygen,hydrogen, and nitrogen. Although they represent a smaller percentage ofthe content of a cell, inorganic substances are equally as important tothe proper functioning of the cell. Such molecules include phosphorous,sulfur, calcium, magnesium, iron, zinc, manganese, copper, molybdenum,tungsten, and cobalt. Many of these compounds are critical for theconstruction of important molecules, such as nucleotides (phosphorous)and amino acids (nitrogen and sulfur). Others of these inorganic ionsserve as cofactors for enzymic reactions or contribute to osmoticpressure. All such molecules must be taken up by the bacterium from thesurrounding environment.

For each of these inorganic compounds it is desirable for the bacteriumto take up the form which can be most readily used by the standardmetabolic machinery of the cell. However, the bacterium may encounterenvironments in which these preferred forms are not readily available.In order to survive under these circumstances, it is important forbacteria to have additional biochemical mechanisms which are able toconvert less metabolically active but readily available forms of theseinorganic compounds to ones which may be used in cellular metabolism.Bacteria frequently possess a number of genes encoding enzymes for thispurpose, which are not expressed unless the desired inorganic speciesare not available. Thus, these genes for the metabolism of variousinorganic compounds serve as another tool which bacteria may use toadapt to suboptimal environmental conditions.

After carbon, the most important element in the cell is nitrogen. Atypical bacterial cell contains between 12-15% nitrogen. It is aconstituent of amino acids and nucleotides, as well as many otherimportant molecules in the cell. Further, nitrogen may serve as asubstitute for oxygen as a terminal electron acceptor in energymetabolism. Good sources of nitrogen include many organic and inorganiccompounds, such ammonia gas or ammonia salts (e.g., NH₄Cl, (NH₄)₂SO₄, orNH₄OH), nitrates, urea, amino acids, or complex nitrogen sources likecorn steep liquor, soy bean flour, soy bean protein, yeast extract, meatextract, etc. Ammonia nitrogen is fixed by the action of particularenzymes: glutamate dehydrogenase, glutamine synthase, andglutamine-2-oxoglutarate aminotransferase. The transfer ofamino-nitrogen from one organic molecule to another is accomplished bythe aminotransferases, a class of enzymes which transfer one amino groupfrom an alpha-amino acid to an alpha-keto acid. Nitrate may be reducedvia nitrate reductase, nitrite reductase, and further redox enzymesuntil it is converted to molecular nitrogen or ammonia, which may bereadily utilized by the cell in standard metabolic pathways.

Phosphorous is typically found intracellularly in both organic andinorganic forms, and may be taken up by the cell in either of theseforms as well, though most microorganisms preferentially take upinorganic phosphate. The conversion of organic phosphate to a form whichthe cell can utilize requires the action of phosphatases (e.g.,phytases, which hydrolyze phyate-yielding phosphate and inositolderivatives). Phosphate is a key element in the synthesis of nucleicacids, and also has a significant role in cellular energy metabolism(e.g., in the synthesis of ATP, ADP, and AMP).

Sulfur is a requirement for the synthesis of amino acids (e.g.,methionine and cysteine), vitamins (e.g., thiamine, biotin, and lipoicacid) and iron sulfur proteins. Bacteria obtain sulfur primarily frominorganic sulfate, though thiosulfate, sulfite, and sulfide are alsocommonly utilized. Under conditions where these compounds may not bereadily available, many bacteria express genes which enable them toutilize sulfonate compounds such as 2-aminosulfonate (taurine) (Kertesz,M. A. (1993) “Proteins induced by sulfate limitation in Escherichiacoli, Pseudomonas putida, or Staphylococcus aureus.” J. Bacteriol. 175:1187-1190).

Other inorganic atoms, e.g., metal or calcium ions, are also criticalfor the viability of cells. Iron, for example, plays a key role in redoxreactions and is a cofactor of iron-sulfur proteins, heme proteins, andcytochromes. The uptake of iron into bacterial cells may be accomplishedby the action of siderophores, chelating agents which bind extracellulariron ions and translocate them to the interior of the cell. Forreference on the metabolism of iron and other inorganic compounds, see:Lengeler et al. (1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart;Neidhardt, F. C. et al., eds. Escherichia coli and Salmonella. ASMPress: Washington, D.C.; Sonenshein, A. L. et al., eds. (199?) Bacillussubtilis and Other Gram-Positive Bacteria, ASM Press: Washington, D.C.;Voet, D. and Voet, J. G. (1992) Biochemie, VCH: Weinheim; Brock, T. D.and Madigan, M. T. (1991) Biology of Microorgansisms, 6^(th) ed.Prentice Hall: Englewood Cliffs, p. 267-269; Rhodes, P. M. and Stanbury,P. F. Applied Microbial Physiology—A Practical Approach, Oxford Univ.Press: Oxford.

C. Enzymes and Proteolysis

The intracellular conditions for which bacteria such as C. glutamicumare optimized are frequently not conditions under which many biochemicalreactions would normally take place. In order to make such reactionsproceed under physiological conditions, cells utilize enzymes. Enzymesare proteinaceous biological catalysts, spatially orienting reactingmolecules or providing a specialized environment such that the energybarrier to a biochemical reaction is lowered. Different enzymes catalyzedifferent reactions, and each enzyme may be the subject oftranscriptional, translational, or posttranslational regulation suchthat the reaction will only take place under appropriate conditions andat specified times. Enzymes may contribute to the degradation (e.g., theproteases), synthesis (e.g., the synthases), or modification (e.g.,transferases or isomerases) of compounds, all of which enable theproduction of necessary compounds within the cell. This, in turn,contributes to the maintenance of cellular homeostasis.

However, the fact that enzymes are optimized for activity under thephysiological conditions at which the bacterium is most viable meansthat when environmental conditions are perturbed, there is a significantpossibility that enzyme activity will also be perturbed. For example,changes in temperature may result in aberrantly folded proteins, and thesame is true for changes of pH—protein folding is largely dependent onelectrostatic and hydrophobic interactions of amino acids within thepolypeptide chain, so any alteration to the charges on individual aminoacids (as might be brought about by a change in cellular pH) may have aprofound effect on the ability of the protein to correctly fold. Changesin temperature effectively change the amount of kinetic energy that thepolypeptide molecule possesses, which affects the ability of thepolypeptide to settle into a correctly folded, energetically stableconfiguration. Misfolded proteins may be harmful to the cell for tworeasons. First, the aberrantly folded protein may have a similarlyaberrant activity, or no activity whatsoever. Second, misfolded proteinsmay lack the conformational regions necessary for proper regulation byother cellular systems and thus may continue to be active but in anuncontrolled fashion.

The cell has a mechanism by which misfolded enzymes and regulatoryproteins may be rapidly destroyed before any damage occurs to the cell:proteolysis. Proteins such as those of the la/Ion family and those ofthe Clp family specifically recognize and degrade misfolded proteins(see, e.g., Sherman, M. Y., Goldberg, A. L. (1999) EXS 77: 57-78 andreferences therein and Porankiewicz J. (1999) Molec. Microbiol. 32(3):449-58, and references therein; Neidhardt, F. C., et al. (1996) E. coliand Salmonella, ASM Press: Washington, D.C. and references therein; andPritchard, G. G., and Coolbear, T. (1993) FEMS Microbiol. Rev. 12(1-3):179-206 and references therein). These enzymes bind to misfolded orunfolded proteins and degrade them in an ATP-dependent manner.Proteolysis thus serves as an important mechanism employed by the cellto prevent damage to normal cellular functions upon environmentalchanges, and it further permits cells to survive under conditions and inenvironments which would otherwise be toxic due to misregulated and/oraberrant enzyme or regulatory activity.

Proteolysis also has important functions in the cell under optimalenvironmental conditions. Within normal metabolic processes, proteasesaid in the hydrolysis of peptide bonds, in the catabolism of complexmolecules to provide necessary degradation products, and in proteinmodification. Secreted proteases play an important role in thecatabolism of external nutrients even prior to the entry of thesecompounds into the cell. Further, proteolytic activity itself may serveregulatory functions; sporulation in B. subtilis and cell cycleprogression in Caulobacter spp. are known to be regulated by keyproteolytic events in each of these species (Gottesman, S. (1999) Curr.Opin. Microbiol. 2(2): 142-147). Thus, proteolytic processes are key forcellular survival under both suboptimal and optimal environmentalconditions, and contribute to the overall maintenance of homeostasis incells.

D. Cell Wall Production and Rearrangements

While the biochemical machinery of the cell may be able to readily adaptto different and possibly unfavorable environments, cells still requirea general mechanism by which they may be protected from the environment.For many bacteria, the cell wall affords such protection, and also playsroles in adhesion, cell growth and division, and transport of desiredsolutes and waste materials.

In order to function, cells require intracellular concentrations ofmetabolites and other molecules that are substantially higher than thoseof the surrounding media. Since these metabolites are largely preventedfrom leaving the cell due to the presence of the hydrophobic membrane,the tendency of the system is for water molecules to enter the cell fromthe external medium such that the interior concentrations of solutesmatch the exterior concentrations. Water molecules are readily able tocross the cellular membrane, and this membrane is not able to withstandthe resulting swelling and pressure, which may lead to osmotic lysis ofthe cell. The rigidity of the cell wall greatly improves the ability ofthe cell to tolerate these pressures, and offers a further barrier tothe unwanted diffusion of these metabolites and desired solutes from thecell. Similarly, the cell wall also serves to prevent unwanted materialfrom entering the cell.

The cell wall also participates in a number of other cellular processes,such as adhesion and cell growth and division. Due to the fact that thecell wall completely surrounds the cell, any interaction of the cellwith its surroundings must be mediated by the cell wall. Thus, the cellwall must participate in any adherence of the cell to other cells and todesired surfaces. Further, the cell cannot grow or divide withoutconcomitant changes in the cell wall. Since the protection that the wallaffords requires its presence during growth, morphogenesis andmultiplication, one of the key steps in cell division is cell wallsynthesis within the cell such that a new cell divides from the old.Thus, frequently cell wall biosynthesis is regulated in tandem with cellgrowth and cell division (see, e.g., Sonenshein, A. L. et al, eds.(1993) Bacillus subtilis and Other Gram-Positive Bacteria, ASM:Washington, D.C.).

The structure of the cell wall varies between gram-positive andgram-negative bacteria. However, in both types, the fundamentalstructural unit of the wall remains similar: an overlapping lattice oftwo polysaccharides, N-acetyl glucosamine (NAG) and N-acetyl muramicacid (NAM) which are cross-linked by amino acids (most commonlyL-alanine, D-glutamate, diaminopimelic acid, and D-alanine), termed‘peptidoglycan’. The processes involved in the synthesis of the cellwall are known (see, e.g., Michal, G., ed. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, Wiley: New York).

In gram-negative bacteria, the inner cellular membrane is coated by asingle-layered peptidoglycan (approximately 10 nm thick), termed themurein-sacculus. This peptidoglycan structure is very rigid, and itsstructure determines the shape of the organism. The outer surface of themurein-sacculus is covered with an outer membrane, containing porins andother membrane proteins, phospholipids, and lipopolysaccharides. Tomaintain a tight association with the outer membrane, the gram-negativecell wall also has interspersed lipid molecules which serve to anchor itto the surrounding membrane.

In gram-positive bacteria, such as Corynebacterium glutamicum, thecytoplasmic membrane is covered by a multi-layered peptidoglycan, whichranges from 20-80 nm in thickness (see, e.g., Lengeler et al. (1999)Biology of Prokaryotes Thieme Verlag: Stuttgart, p. 913-918, p. 875-899,and p. 88-109 and references therein). The gram-positive cell wall alsocontains teichoic acid, a polymer of glycerol or ribitol linked throughphosphate groups. Teichoic acid is also able to associate with aminoacids, and forms covalent bonds with muramic acid. Also present in thecell wall may be lipoteichoic acids and teichuronic acids. If present,cellular surface structures such as flagella or capsules will beanchored in this layer as well.

III. Elements and Methods of the Invention

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as HA nucleic acid and proteinmolecules, which participate in the maintenance of homeostasis in C.glutamicum, or which perform a function involved in the adaptation ofthis microorganism to different environmental conditions. In oneembodiment, the HA molecules participate in C. glutamicum cell wallbiosynthesis or rearrangements, in the metabolism of inorganiccompounds, in the modification or degradation of aromatic or aliphaticcompounds, or have an enzymatic or proteolytic activity. In a preferredembodiment, the activity of the HA molecules of the present inventionwith regard to C. glutamicum cell wall biosynthesis or rearrangements,metabolism of inorganic compounds, modification or degradation ofaromatic or aliphatic compounds, or enzymatic or proteolytic activityhas an impact on the production of a desired fine chemical by thisorganism. In a particularly preferred embodiment, the HA molecules ofthe invention are modulated in activity, such that the C. glutamicumcellular processes in which the HA molecules participate (e.g., C.glutamicum cell wall biosynthesis or rearrangements, metabolism ofinorganic compounds, modification or degradation of aromatic oraliphatic compounds, or enzymatic or proteolytic activity) are alsoaltered in activity, resulting either directly or indirectly in amodulation of the yield, production, and/or efficiency of production ofa desired fine chemical by C. glutamicum.

The language, “HA protein” or “HA polypeptide” includes proteins whichparticipate in a number of cellular processes related to C. glutamicumhomeostasis or the ability of C. glutamicum cells to adapt tounfavorable environmental conditions. For example, an HA protein may beinvolved in C. glutamicum cell wall biosynthesis or rearrangements, inthe metabolism of inorganic compounds in C. glutamicum, in themodification or degradation of aromatic or aliphatic compounds in C.glutamicum, or have a C. glutamicum enzymatic or proteolytic activity.Examples of HA proteins include those encoded by the HA genes set forthin Table 1 and Appendix A. The terms “HA gene” or “HA nucleic acidsequence” include nucleic acid sequences encoding an HA protein, whichconsist of a coding region and also corresponding untranslated 5′ and 3′sequence regions. Examples of HA genes include those set forth inTable 1. The terms “production” or “productivity” are art-recognized andinclude the concentration of the fermentation product (for example, thedesired fine chemical) formed within a given time and a givenfermentation volume (e.g., kg product per hour per liter). The term“efficiency of production” includes the time required for a particularlevel of production to be achieved (for example, how long it takes forthe cell to attain a particular rate of output of a fine chemical). Theterm “yield” or “product/carbon yield” is art-recognized and includesthe efficiency of the conversion of the carbon source into the product(i.e., fine chemical). This is generally written as, for example, kgproduct per kg carbon source. By increasing the yield or production ofthe compound, the quantity of recovered molecules, or of usefulrecovered molecules of that compound in a given amount of culture over agiven amount of time is increased. The terms “biosynthesis” or a“biosynthetic pathway” are art-recognized and include the synthesis of acompound, preferably an organic compound, by a cell from intermediatecompounds in what may be a multistep and highly regulated process. Theterms “degradation” or a “degradation pathway” are art-recognized andinclude the breakdown of a compound, preferably an organic compound, bya cell to degradation products (generally speaking, smaller or lesscomplex molecules) in what may be a multistep and highly regulatedprocess. The language “metabolism” is art-recognized and includes thetotality of the biochemical reactions that take place in an organism.The metabolism of a particular compound, then, (e.g., the metabolism ofan amino acid such as glycine) comprises the overall biosynthetic,modification, and degradation pathways in the cell related to thiscompound. The term “homeostasis” is art-recognized and includes all ofthe mechanisms utilized by a cell to maintain a constant intracellularenvironment despite the prevailing extracellular environmentalconditions. A non-limiting example of such processes is the utilizationof a cell wall to prevent osmotic lysis due to high intracellular soluteconcentrations. The term “adaptation” or “adaptation to an environmentalcondition” is art-recognized and includes mechanisms utilized by thecell to render the cell able to survive under nonpreferred environmentalconditions (generally speaking, those environmental conditions in whichone or more favored nutrients are absent, or in which an environmentalcondition such as temperature, pH, osmolarity, oxygen percentage and thelike fall outside of the optimal survival range of the cell). Manycells, including C. glutamicum cells, possess genes encoding proteinswhich are expressed under such environmental conditions and which permitcontinued growth in such suboptimal conditions.

In another embodiment, the HA molecules of the invention are capable ofmodulating the production of a desired molecule, such as a finechemical, in a microorganism such as C. glutamicum. There are a numberof mechanisms by which the alteration of an HA protein of the inventionmay directly affect the yield, production, and/or efficiency ofproduction of a fine chemical from a C. glutamicum strain incorporatingsuch an altered protein. For example, by engineering enzymes whichmodify or degrade aromatic or aliphatic compounds such that theseenzymes are increased or decreased in activity or number, it may bepossible to modulate the production of one or more fine chemicals whichare the modification or degradation products of these compounds.Similarly, enzymes involved in the metabolism of inorganic compoundsprovide key molecules (e.g. phosphorous, sulfur, and nitrogen molecules)for the biosynthesis of such fine chemicals as amino acids, vitamins,and nucleic acids. By altering the activity or number of these enzymesin C. glutamicum, it may be possible to increase the conversion of theseinorganic compounds (or to use alternate inorganic compounds) to thuspermit improved rates of incorporation of inorganic atoms into thesefine chemicals. Genetic engineering of C. glutamicum enzymes involved ingeneral cellular processes may also directly improve fine chemicalproduction, since many of these enzymes directly modify fine chemicals(e.g., amino acids) or the enzymes which are involved in fine chemicalsynthesis or secretion. Modulation of the activity or number of cellularproteases may also have a direct effect on fine chemical production,since many proteases may degrade fine chemicals or enzymes involved infine chemical production or breakdown.

Further, the aforementioned enzymes which participate inaromatic/aliphatic compound modification or degradation, generalbiocatalysis, inorganic compound metabolism or proteolysis are eachthemselves fine chemicals, desirable for their activity in various invitro industrial applications. By altering the number of copies of thegene for one or more of these enzymes in C. glutamicum it may bepossible to increase the number of these proteins produced by the cell,thereby increasing the potential yield or efficiency of production ofthese proteins from large-scale C. glutamicum or related bacterialcultures.

The alteration of an HA protein of the invention may also indirectlyaffect the yield, production, and/or efficiency of production of a finechemical from a C. glutamicum strain incorporating such an alteredprotein. For example, by modulating the activity and/or number of thoseproteins involved in the construction or rearrangement of the cell wall,it may be possible to modify the structure of the cell wall itself suchthat the cell is able to better withstand the mechanical and otherstresses present during large-scale fermentative culture. Also,large-scale growth of C. glutamicum requires significant cell wallproduction. Modulation of the activity or number of cell wallbiosynthetic or degradative enzymes may allow more rapid rates of cellwall biosynthesis, which in turn may permit increased growth rates ofthis microorganism in culture and thereby increase the number of cellsproducing the desired fine chemical.

By modifying the HA enzymes of the invention, one may also indirectlyimpact the yield, production, or efficiency of production of one or morefine chemicals from C. glutamicum. For example, many of the generalenzymes in C. glutamicum may have a significant impact on globalcellular processes (e.g., regulatory processes) which in turn have asignificant effect on fine chemical metabolism. Similarly, proteases,enzymes which modify or degrade possibly toxic aromatic or aliphaticcompounds, and enzymes which promote the metabolism of inorganiccompounds all serve to increase the viability of C. glutamicum. Theproteases aid in the selective removal of misfolded or misregulatedproteins, such as those that might occur under the relatively stressfulenvironmental conditions encountered during large-scale fermentorculture. By altering these proteins, it may be possible to furtherenhance this activity and to improve the viability of C. glutamicum inculture. The aromatic/aliphatic modification or degradation proteins notonly serve to detoxify these waste compounds (which may be encounteredas impurities in culture medium or as waste products from cellsthemselves), but also to permit the cells to utilize alternate carbonsources if the optimal carbon source is limiting in the culture. Byincreasing their number and/or activity, the survival of C. glutamicumcells in culture may be enhanced. The inorganic metabolism proteins ofthe invention supply the cell with inorganic molecules required for allprotein and nucleotide (among others) synthesis, and thus are criticalfor the overall viability of the cell. An increase in the number ofviable cells producing one or more desired fine chemicals in large-scaleculture should result in a concomitant increase in the yield,production, and/or efficiency of production of the fine chemical in theculture.

The isolated nucleic acid sequences of the invention are containedwithin the genome of a Corynebacterium glutamicum strain availablethrough the American Type Culture Collection, given designation ATCC13032. The nucleotide sequence of the isolated C. glutamicum HA DNAs andthe predicted amino acid sequences of the C. glutamicum HA proteins areshown in Appendices A and B, respectively. Computational analyses wereperformed which classified and/or identified these nucleotide sequencesas sequences which encode proteins that participate in C. glutamicumcell wall biosynthesis or rearrangements, metabolism of inorganiccompounds, modification or degradation of aromatic or aliphaticcompounds, or that have a C. glutamicum enzymatic or proteolyticactivity.

The present invention also pertains to proteins which have an amino acidsequence which is substantially homologous to an amino acid sequence ofAppendix B. As used herein, a protein which has an amino acid sequencewhich is substantially homologous to a selected amino acid sequence isleast about 50% homologous to the selected amino acid sequence, e.g.,the entire selected amino acid sequence. A protein which has an aminoacid sequence which is substantially homologous to a selected amino acidsequence can also be least about 50-60%, preferably at least about60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%,and most preferably at least about 96%, 97%, 98%, 99% or more homologousto the selected amino acid sequence.

The HA protein or a biologically active portion or fragment thereof ofthe invention can participate in the maintenance of homeostasis in C.glutamicum, or can perform a function involved in the adaptation of thismicroorganism to different environmental conditions, or have one or moreof the activities set forth in Table 1.

Various aspects of the invention are described in further detail in thefollowing subsections.

A. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode HA polypeptides or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes or primers for the identification or amplification of HA-encodingnucleic acid (e.g., HA DNA). As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 100 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 20 nucleotidesof sequence downstream from the 3′end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolated HAnucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived (e.g., a C. glutamicum cell). Moreover, an“isolated” nucleic acid molecule, such as a DNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of Appendix A, or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. For example, a C. glutamicumHA DNA can be isolated from a C. glutamicum library using all or portionof one of the sequences of Appendix A as a hybridization probe andstandard hybridization techniques (e.g., as described in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acidmolecule encompassing all or a portion of one of the sequences ofAppendix A can be isolated by the polymerase chain reaction usingoligonucleotide primers designed based upon this sequence (e.g., anucleic acid molecule encompassing all or a portion of one of thesequences of Appendix A can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this same sequence ofAppendix A). For example, mRNA can be isolated from normal endothelialcells (e.g., by the guanidinium-thiocyanate extraction procedure ofChirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA can beprepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for polymerase chain reactionamplification can be designed based upon one of the nucleotide sequencesshown in Appendix A. A nucleic acid of the invention can be amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to an HA nucleotide sequencecan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in Appendix A.The sequences of Appendix A correspond to the Corynebacterium glutamicumHA DNAs of the invention. This DNA comprises sequences encoding HAproteins (i.e., the “coding region”, indicated in each sequence inAppendix A), as well as 5′ untranslated sequences and 3′ untranslatedsequences, also indicated in Appendix A. Alternatively, the nucleic acidmolecule can comprise only the coding region of any of the sequences inAppendix A.

For the purposes of this application, it will be understood that each ofthe sequences set forth in Appendix A has an identifying RXA, RXN, RXS,or RXC number having the designation “RXA,” “RXN,” “RXS, or “RXC”followed by 5 digits (i.e., RXA02458, RXN00249, RXS00153, or RXC00963).Each of these sequences comprises up to three parts: a 5′ upstreamregion, a coding region, and a downstream region. Each of these threeregions is identified by the same RXA, RXN, RXS, or RXC designation toeliminate confusion. The recitation “one of the sequences in AppendixA”, then, refers to any of the sequences in Appendix A, which may bedistinguished by their differing RXA, RXN, RXS, or RXC designations. Thecoding region of each of these sequences is translated into acorresponding amino acid sequence, which is set forth in Appendix B. Thesequences of Appendix B are identified by the same RXA, RXN, RXS, or RXCdesignations as Appendix A, such that they can be readily correlated.For example, the amino acid sequences in Appendix B designated RXA02458,RXN00249, RXS00153, and RXC00963 are translations of the coding regionsof the nucleotide sequences of nucleic acid molecules RXA02458,RXN00249, RXS00153, and RXC00963, respectively, in Appendix A. Each ofthe RXA, RXN, RXS, and RXC nucleotide and amino acid sequences of theinvention has also been assigned a SEQ ID NO, as indicated in Table 1.

Several of the genes of the invention are “F-designated genes”. AnF-designated gene includes those genes set forth in Table 1 which havean ‘F’ in front of the RXA, RXN, RXS, or RXC designation. For example,SEQ ID NO:5, designated, as indicated on Table 1, as “F RXA00249”, is anF-designated gene, as are SEQ ID NOs: 11, 15, and 33 (designated onTable 1 as “F RXA02264”, “F RXA02274”, and “F RXA00675”, respectively).

In one embodiment, the nucleic acid molecules of the present inventionare not intended to include those compiled in Table 2. In the case ofthe dapD gene, a sequence for this gene was published in Wehrmann, A.,et al. (1998) J. Bacteriol. 180(12): 3159-3165. However, the sequenceobtained by the inventors of the present application is significantlylonger than the published version. It is believed that the publishedversion relied on an incorrect start codon, and thus represents only afragment of the actual coding region.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofone of the nucleotide sequences shown in Appendix A, or a portionthereof. A nucleic acid molecule which is complementary to one of thenucleotide sequences shown in Appendix A is one which is sufficientlycomplementary to one of the nucleotide sequences shown in Appendix Asuch that it can hybridize to one of the nucleotide sequences shown inAppendix A, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleotide sequence which is at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably atleast about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, morepreferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%,92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,98%, 99% or more homologous to a nucleotide sequence shown in AppendixA, or a portion thereof. Ranges and identity values intermediate to theabove-recited ranges, (e.g., 70-90% identical or 80-95% identical) arealso intended to be encompassed by the present invention. For example,ranges of identity values using a combination of any of the above valuesrecited as upper and/or lower limits are intended to be included. In anadditional preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to one of the nucleotidesequences shown in Appendix A, or a portion thereof.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in Appendix A, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of an HA protein. The nucleotidesequences determined from the cloning of the HA genes from C. glutamicumallows for the generation of probes and primers designed for use inidentifying and/or cloning HA homologues in other cell types andorganisms, as well as HA homologues from other Corynebacteria or relatedspecies. The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of a sense strand of one of the sequences setforth in Appendix A, an anti-sense sequence of one of the sequences setforth in Appendix A, or naturally occurring mutants thereof. Primersbased on a nucleotide sequence of Appendix A can be used in PCRreactions to clone HA homologues. Probes based on the HA nucleotidesequences can be used to detect transcripts or genomic sequencesencoding the same or homologous proteins. In preferred embodiments, theprobe further comprises a label group attached thereto, e.g. the labelgroup can be a radioisotope, a fluorescent compound, an enzyme, or anenzyme co-factor. Such probes can be used as a part of a diagnostic testkit for identifying cells which misexpress an HA protein, such as bymeasuring a level of an HA-encoding nucleic acid in a sample of cells,e.g., detecting HA mRNA levels or determining whether a genomic HA genehas been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of Appendix B suchthat the protein or portion thereof maintains the ability to participatein the maintenance of homeostasis in C. glutamicum, or to perform afunction involved in the adaptation of this microorganism to differentenvironmental conditions. As used herein, the language “sufficientlyhomologous” refers to proteins or portions thereof which have amino acidsequences which include a minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain as an aminoacid residue in one of the sequences of Appendix B) amino acid residuesto an amino acid sequence of Appendix B such that the protein or portionthereof is able to participate in the maintenance of homeostasis in C.glutamicum, or to perform a function involved in the adaptation of thismicroorganism to different environmental conditions. Proteins involvedin C. glutamicum cell wall biosynthesis or rearrangements, metabolism ofinorganic compounds, modification or degradation of aromatic oraliphatic compounds, or that have a C. glutamicum enzymatic orproteolytic activity, as described herein, may play a role in theproduction and secretion of one or more fine chemicals. Examples of suchactivities are also described herein. Thus, “the function of an HAprotein” contributes either directly or indirectly to the yield,production, and/or efficiency of production of one or more finechemicals. Examples of HA protein activities are set forth in Table 1.

In another embodiment, the protein is at least about 50-60%, preferablyat least about 60-70%, and more preferably at least about 70-80%,80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% ormore homologous to an entire amino acid sequence of Appendix B.

Portions of proteins encoded by the HA nucleic acid molecules of theinvention are preferably biologically active portions of one of the HAproteins. As used herein, the term “biologically active portion of an HAprotein” is intended to include a portion, e.g., a domain/motif, of anHA protein that can participate in the maintenance of homeostasis in C.glutamicum, or that can perform a function involved in the adaptation ofthis microorganism to different environmental conditions, or has anactivity as set forth in Table 1. To determine whether an HA protein ora biologically active portion thereof can participate in C. glutamicumcell wall biosynthesis or rearrangements, metabolism of inorganiccompounds, modification or degradation of aromatic or aliphaticcompounds, or has a C. glutamicum enzymatic or proteolytic activity, anassay of enzymatic activity may be performed. Such assay methods arewell known to those of ordinary skill in the art, as detailed in Example8 of the Exemplification.

Additional nucleic acid fragments encoding biologically active portionsof an HA protein can be prepared by isolating a portion of one of thesequences in Appendix B, expressing the encoded portion of the HAprotein or peptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the HA protein orpeptide.

The invention further encompasses nucleic acid molecules that differfrom one of the nucleotide sequences shown in Appendix A (and portionsthereof) due to degeneracy of the genetic code and thus encode the sameHA protein as that encoded by the nucleotide sequences shown in AppendixA. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence shown in Appendix B. In a still further embodiment, thenucleic acid molecule of the invention encodes a full length C.glutamicum protein which is substantially homologous to an amino acidsequence of Appendix B (encoded by an open reading frame shown inAppendix A).

It will be understood by one of ordinary skill in the art that in oneembodiment the sequences of the invention are not meant to include thesequences of the prior art, such as those Genbank sequences set forth inTables 2 or 4 which were available prior to the present invention. Inone embodiment, the invention includes nucleotide and amino acidsequences having a percent identity to a nucleotide or amino acidsequence of the invention which is greater than that of a sequence ofthe prior art (e.g., a Genbank sequence (or the protein encoded by sucha sequence) set forth in Tables 2 or 4). For example, the inventionincludes a nucleotide sequence which is greater than and/or at least 39%identical to the nucleotide sequence designated RXA00471 (SEQ IDNO:293), a nucleotide sequence which is greater than and/or at least 41%identical to the nucleotide sequence designated RXA00500 (SEQ IDNO:143), and a nucleotide sequence which is greater than and/or at least35% identical to the nucleotide sequence designated RXA00502 (SEQ IDNO:147). One of ordinary skill in the art would be able to calculate thelower threshold of percent identity for any given sequence of theinvention by examining the GAP-calculated percent identity scores setforth in Table 4 for each of the three top hits for the given sequence,and by subtracting the highest GAP-calculated percent identity from 100percent. One of ordinary skill in the art will also appreciate thatnucleic acid and amino acid sequences having percent identities greaterthan the lower threshold so calculated (e.g., at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably atleast about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%,and even more preferably at least about 95%, 96%, 97%, 98%, 99% or moreidentical) are also encompassed by the invention.

In addition to the C. glutamicum HA nucleotide sequences shown inAppendix A, it will be appreciated by those of ordinary skill in the artthat DNA sequence polymorphisms that lead to changes in the amino acidsequences of HA proteins may exist within a population (e.g., the C.glutamicum population). Such genetic polymorphism in the HA gene mayexist among individuals within a population due to natural variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding an HA protein,preferably a C. glutamicum HA protein. Such natural variations cantypically result in 1-5% variance in the nucleotide sequence of the HAgene. Any and all such nucleotide variations and resulting amino acidpolymorphisms in HA that are the result of natural variation and that donot alter the functional activity of HA proteins are intended to bewithin the scope of the invention.

Nucleic acid molecules corresponding to natural variants and non-C.glutamicum homologues of the C. glutamicum HA DNA of the invention canbe isolated based on their homology to the C. glutamicum HA nucleic aciddisclosed herein using the C. glutamicum DNA, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. Accordingly, in another embodiment,an isolated nucleic acid molecule of the invention is at least 15nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising a nucleotide sequence of Appendix A. Inother embodiments, the nucleic acid is at least 30, 50, 100, 250 or morenucleotides in length. As used herein, the term “hybridizes understringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%homologous to each other typically remain hybridized to each other.Preferably, the conditions are such that sequences at least about 65%,more preferably at least about 70%, and even more preferably at leastabout 75% or more homologous to each other typically remain hybridizedto each other. Such stringent conditions are known to those of ordinaryskill in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred,non-limiting example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to a sequence of Appendix Acorresponds to a naturally-occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein). In one embodiment, the nucleic acidencodes a natural C. glutamicum HA protein.

In addition to naturally-occurring variants of the HA sequence that mayexist in the population, one of ordinary skill in the art will furtherappreciate that changes can be introduced by mutation into a nucleotidesequence of Appendix A, thereby leading to changes in the amino acidsequence of the encoded HA protein, without altering the functionalability of the HA protein. For example, nucleotide substitutions leadingto amino acid substitutions at “non-essential” amino acid residues canbe made in a sequence of Appendix A. A “non-essential” amino acidresidue is a residue that can be altered from the wild-type sequence ofone of the HA proteins (Appendix B) without altering the activity ofsaid HA protein, whereas an “essential” amino acid residue is requiredfor HA protein activity. Other amino acid residues, however, (e.g.,those that are not conserved or only semi-conserved in the domain havingHA activity) may not be essential for activity and thus are likely to beamenable to alteration without altering HA activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding HA proteins that contain changes in amino acidresidues that are not essential for HA activity. Such HA proteins differin amino acid sequence from a sequence contained in Appendix B yetretain at least one of the HA activities described herein. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein comprises an amino acidsequence at least about 50% homologous to an amino acid sequence ofAppendix B and is capable of participating in the maintenance ofhomeostasis in C. glutamicum, or of performing a function involved inthe adaptation of this microorganism to different environmentalconditions, or has one or more of the activities set forth in Table 1.Preferably, the protein encoded by the nucleic acid molecule is at leastabout 50-60% homologous to one of the sequences in Appendix B, morepreferably at least about 60-70% homologous to one of the sequences inAppendix B, even more preferably at least about 70-80%, 80-90%, 90-95%homologous to one of the sequences in Appendix B, and most preferably atleast about 96%, 97%, 98%, or 99% homologous to one of the sequences inAppendix B.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of Appendix B and a mutant form thereof) or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of one protein or nucleicacid for optimal alignment with the other protein or nucleic acid). Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in onesequence (e.g., one of the sequences of Appendix B) is occupied by thesame amino acid residue or nucleotide as the corresponding position inthe other sequence (e.g., a mutant form of the sequence selected fromAppendix B), then the molecules are homologous at that position (i.e.,as used herein amino acid or nucleic acid “homology” is equivalent toamino acid or nucleic acid “identity”). The percent homology between thetwo sequences is a function of the number of identical positions sharedby the sequences (i.e., % homology=#of identical positions/total #ofpositions×100).

An isolated nucleic acid molecule encoding an HA protein homologous to aprotein sequence of Appendix B can be created by introducing one or morenucleotide substitutions, additions or deletions into a nucleotidesequence of Appendix A such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into one of the sequences of Appendix A bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an HA protein is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of an HA coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened foran HA activity described herein to identify mutants that retain HAactivity. Following mutagenesis of one of the sequences of Appendix A,the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined using, for example, assays describedherein (see Example 8 of the Exemplification).

In addition to the nucleic acid molecules encoding HA proteins describedabove, another aspect of the invention pertains to isolated nucleic acidmolecules which are antisense thereto. An “antisense” nucleic acidcomprises a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded DNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire HA coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding an HAprotein. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acid residues(e.g., the entire coding region of SEQ ID NO. 3 (RXN00249) comprisesnucleotides 1 to 957). In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding HA. The term “noncoding region” refers to5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding HA disclosed herein (e.g.,the sequences set forth in Appendix A), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of HA mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of HA mRNA. For example, the antisense oligonucleotidecan be complementary to the region surrounding the translation startsite of HA mRNA. An antisense oligonucleotide can be, for example, about5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an HA proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic promoter arepreferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual P-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveHA mRNA transcripts to thereby inhibit translation of HA mRNA. Aribozyme having specificity for an HA-encoding nucleic acid can bedesigned based upon the nucleotide sequence of an HA DNA moleculedisclosed herein (i.e., SEQ ID NO. 3 (RXN00249) Appendix A). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in an HA-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.5,116,742. Alternatively, HA mRNA can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, HA gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of an HAnucleotide sequence (e.g., an HA promoter and/or enhancers) to formtriple helical structures that prevent transcription of an HA gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14(12):807-15.

B. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an HA protein (ora portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which are operatively linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells. Preferred regulatory sequences are, for example,promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-,lpp-lac-, lacI^(q), T7-, T5-, T3-, gal-, trc-, ara-, SP6—, arny, SPO2,λ-P_(R)- or λP_(L), which are used preferably in bacteria. Additionalregulatory sequences are, for example, promoters from yeasts and fungi,such as ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, promoters fromplants such as CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos orubiquitin- or phaseolin-promoters. It is also possible to use artificialpromoters. It will be appreciated by those of ordinary skill in the artthat the design of the expression vector can depend on such factors asthe choice of the host cell to be transformed, the level of expressionof protein desired, etc. The expression vectors of the invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein (e.g., HA proteins, mutant forms of HA proteins, fusionproteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of HA proteins in prokaryotic or eukaryotic cells. Forexample, HA genes can be expressed in bacterial cells such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastand other fungal cells (see Romanos, M. A. et al. (1992) “Foreign geneexpression in yeast: a review”, Yeast 8: 423-488; van den Hondel, C. A.M. J. J. et al. (1991) “Heterologous gene expression in filamentousfungi” in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel,C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge University Press:Cambridge), algae and multicellular plant cells (see Schmidt, R. andWillmitzer, L. (1988) High efficiency Agrobacterium tumefaciens—mediatedtransformation of Arabidopsis thaliana leaf and cotyledon explants”Plant Cell Rep.: 583-586), or mammalian cells. Suitable host cells arediscussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the HA protein is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant HA proteinunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) pLG338, pACYC184,pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200,pUR290, pIN-III113-B1, λgt11, pBdCl, and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 60-89; and Pouwels et al., eds. (1985) CloningVectors. Elsevier: New York IBSN 0 444 904018). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter. For transformation of other varietiesof bacteria, appropriate vectors may be selected. For example, theplasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful intransforming Streptomyces, while plasmids pUB110, pC194, or pBD214 aresuited for transformation of Bacillus species. Several plasmids of usein the transfer of genetic information into Corynebacterium includepHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds. (1985) CloningVectors. Elsevier: New York IBSN 0 444 904018).

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in the bacterium chosen for expression, such asC. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118).Such alteration of nucleic acid sequences of the invention can becarried out by standard DNA synthesis techniques.

In another embodiment, the HA protein expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234),2μ, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982)Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), andpYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methodsfor the construction of vectors appropriate for use in other fungi, suchas the filamentous fungi, include those detailed in: van den Hondel, C.A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press:Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier:New York (IBSN 0 444 904018).

Alternatively, the HA proteins of the invention can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In another embodiment, the HA proteins of the invention may be expressedin unicellular plant cells (such as algae) or in plant cells from higherplants (e.g., the spermatophytes, such as crop plants). Examples ofplant expression vectors include those detailed in: Becker, D., Kemper,E., Schell, J. and Masterson, R. (1992) “New plant binary vectors withselectable markers located proximal to the left border”, Plant Mol.Biol. 20: 1195-1197; and Bevan, M. W. (1984) “Binary Agrobacteriumvectors for plant transformation”, Nucl. Acid. Res. 12: 8711-8721, andinclude pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al.,eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Baneiji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to HA mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) (1986).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, anHA protein can be expressed in bacterial cells such as C. glutamicum,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseof ordinary skill in the art. Microorganisms related to Corynebacteriumglutamicum which may be conveniently used as host cells for the nucleicacid and protein molecules of the invention are set forth in Table 3.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., linear DNA or RNA(e.g., a linearized vector or a gene construct alone without a vector)or nucleic acid in the form of a vector (e.g., a plasmid, phage,phasmid, phagemid, transposon or other DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding an HA protein or can be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by, for example, drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

To create a homologous recombinant microorganism, a vector is preparedwhich contains at least a portion of an HA gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the HA gene. Preferably, this HA gene is aCorynebacterium glutamicum HA gene, but it can be a homologue from arelated bacterium or even from a mammalian, yeast, or insect source. Ina preferred embodiment, the vector is designed such that, uponhomologous recombination, the endogenous HA gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a “knock out” vector). Alternatively, the vector can be designedsuch that, upon homologous recombination, the endogenous HA gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous HA protein). In the homologousrecombination vector, the altered portion of the HA gene is flanked atits 5′ and 3′ ends by additional nucleic acid of the HA gene to allowfor homologous recombination to occur between the exogenous HA genecarried by the vector and an endogenous HA gene in a microorganism. Theadditional flanking HA nucleic acid is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see e.g., Thomas, K. R., and Capecchi, M. R.(1987) Cell 51: 503 for a description of homologous recombinationvectors). The vector is introduced into a microorganism (e.g., byelectroporation) and cells in which the introduced HA gene hashomologously recombined with the endogenous HA gene are selected, usingart-known techniques.

In another embodiment, recombinant microorganisms can be produced whichcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of an HA gene on a vectorplacing it under control of the lac operon permits expression of the HAgene only in the presence of IPTG. Such regulatory systems are wellknown in the art.

In another embodiment, an endogenous HA gene in a host cell is disrupted(e.g., by homologous recombination or other genetic means known in theart) such that expression of its protein product does not occur. Inanother embodiment, an endogenous or introduced HA gene in a host cellhas been altered by one or more point mutations, deletions, orinversions, but still encodes a functional HA protein. In still anotherembodiment, one or more of the regulatory regions (e.g., a promoter,repressor, or inducer) of an HA gene in a microorganism has been altered(e.g., by deletion, truncation, inversion, or point mutation) such thatthe expression of the HA gene is modulated. One of ordinary skill in theart will appreciate that host cells containing more than one of thedescribed HA gene and protein modifications may be readily producedusing the methods of the invention, and are meant to be included in thepresent invention.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an HA protein.Accordingly, the invention further provides methods for producing HAproteins using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding an HA protein has beenintroduced, or into which genome has been introduced a gene encoding awild-type or altered HA protein) in a suitable medium until HA proteinis produced. In another embodiment, the method further comprisesisolating HA proteins from the medium or the host cell.

C. Isolated HA Proteins

Another aspect of the invention pertains to isolated HA proteins, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is substantially free ofcellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof HA protein in which the protein is separated from cellular componentsof the cells in which it is naturally or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of HA protein having less than about 30% (by dryweight) of non-HA protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-HA protein, stillmore preferably less than about 10% of non-HA protein, and mostpreferably less than about 5% non-HA protein. When the HA protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation. The language “substantially free of chemical precursors orother chemicals” includes preparations of HA protein in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of HA protein having less than about 30% (by dryweight) of chemical precursors or non-HA chemicals, more preferably lessthan about 20% chemical precursors or non-HA chemicals, still morepreferably less than about 10% chemical precursors or non-HA chemicals,and most preferably less than about 5% chemical precursors or non-HAchemicals. In preferred embodiments, isolated proteins or biologicallyactive portions thereof lack contaminating proteins from the sameorganism from which the HA protein is derived. Typically, such proteinsare produced by recombinant expression of, for example, a C. glutamicumHA protein in a microorganism such as C. glutamicum.

An isolated HA protein or a portion thereof of the invention canparticipate in the repair or recombination of DNA, in the transpositionof genetic material, in gene expression (i.e., the processes oftranscription or translation), in protein folding, or in proteinsecretion in Corynebacterium glutamicum, or has one or more of theactivities set forth in Table 1. In preferred embodiments, the proteinor portion thereof comprises an amino acid sequence which issufficiently homologous to an amino acid sequence of Appendix B suchthat the protein or portion thereof maintains the ability to participatein the maintenance of homeostasis in C. glutamicum, or to perform afunction involved in the adaptation of this microorganism to differentenvironmental conditions. The portion of the protein is preferably abiologically active portion as described herein. In another preferredembodiment, an HA protein of the invention has an amino acid sequenceshown in Appendix B. In yet another preferred embodiment, the HA proteinhas an amino acid sequence which is encoded by a nucleotide sequencewhich hybridizes, e.g., hybridizes under stringent conditions, to anucleotide sequence of Appendix A. In still another preferredembodiment, the HA protein has an amino acid sequence which is encodedby a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at leastabout 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, andeven more preferably at least about 95%, 96%, 97%, 98%, 99% % or morehomologous to one of the nucleic acid sequences of Appendix A, or aportion thereof. Ranges and identity values intermediate to theabove-recited values, (e.g., 70-90% identical or 80-95% identical) arealso intended to be encompassed by the present invention. For example,ranges of identity values using a combination of any of the above valuesrecited as upper and/or lower limits are intended to be included. Thepreferred HA proteins of the present invention also preferably possessat least one of the HA activities described herein. For example, apreferred HA protein of the present invention includes an amino acidsequence encoded by a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to a nucleotide sequence ofAppendix A, and which can participate in the maintenance of homeostasisin C. glutamicum, or can perform a function involved in the adaptationof this microorganism to different environmental conditions, or whichhas one or more of the activities set forth in Table 1.

In other embodiments, the HA protein is substantially homologous to anamino acid sequence of Appendix B and retains the functional activity ofthe protein of one of the sequences of Appendix B yet differs in aminoacid sequence due to natural variation or mutagenesis, as described indetail in subsection I above. Accordingly, in another embodiment, the HAprotein is a protein which comprises an amino acid sequence which is atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%,preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%,96%, 97%, 98%, 99% or more homologous to an entire amino acid sequenceof Appendix B and which has at least one of the HA activities describedherein. Ranges and identity values intermediate to the above-recitedvalues, (e.g., 70-90% identical or 80-95% identical) are also intendedto be encompassed by the present invention. For example, ranges ofidentity values using a combination of any of the above values recitedas upper and/or lower limits are intended to be included. In anotherembodiment, the invention pertains to a full length C. glutamicumprotein which is substantially homologous to an entire amino acidsequence of Appendix B.

Biologically active portions of an HA protein include peptidescomprising amino acid sequences derived from the amino acid sequence ofan HA protein, e.g., the an amino acid sequence shown in Appendix B orthe amino acid sequence of a protein homologous to an HA protein, whichinclude fewer amino acids than a full length HA protein or the fulllength protein which is homologous to an HA protein, and exhibit atleast one activity of an HA protein. Typically, biologically activeportions (peptides, e.g., peptides which are, for example, 5, 10, 15,20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length)comprise a domain or motif with at least one activity of an HA protein.Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the activities described herein.Preferably, the biologically active portions of an HA protein includeone or more selected domains/motifs or portions thereof havingbiological activity.

HA proteins are preferably produced by recombinant DNA techniques. Forexample, a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the HA protein isexpressed in the host cell. The HA protein can then be isolated from thecells by an appropriate purification scheme using standard proteinpurification techniques. Alternative to recombinant expression, an HAprotein, polypeptide, or peptide can be synthesized chemically usingstandard peptide synthesis techniques. Moreover, native HA protein canbe isolated from cells (e.g., endothelial cells), for example using ananti-HA antibody, which can be produced by standard techniques utilizingan HA protein or fragment thereof of this invention.

The invention also provides HA chimeric or fusion proteins. As usedherein, an HA “chimeric protein” or “fusion protein” comprises an HApolypeptide operatively linked to a non-HA polypeptide. An “HApolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to an HA protein, whereas a “non-HA polypeptide” refers toa polypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the HA protein, e.g., a proteinwhich is different from the HA protein and which is derived from thesame or a different organism. Within the fusion protein, the term“operatively linked” is intended to indicate that the HA polypeptide andthe non-HA polypeptide are fused in-frame to each other. The non-HApolypeptide can be fused to the N-terminus or C-terminus of the HApolypeptide. For example, in one embodiment the fusion protein is aGST-HA fusion protein in which the HA sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant HA proteins. In another embodiment, thefusion protein is an HA protein containing a heterologous signalsequence at its N-terminus. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of an HA protein can be increasedthrough use of a heterologous signal sequence.

Preferably, an HA chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). An HA-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the HA protein.

Homologues of the HA protein can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the HA protein. As used herein,the term “homologue” refers to a variant form of the HA protein whichacts as an agonist or antagonist of the activity of the HA protein. Anagonist of the HA protein can retain substantially the same, or asubset, of the biological activities of the HA protein. An antagonist ofthe HA protein can inhibit one or more of the activities of thenaturally occurring form of the HA protein, by, for example,competitively binding to a downstream or upstream member of abiochemical cascade which includes the HA protein, by binding to atarget molecule with which the HA protein interacts, such that nofunctional interaction is possible, or by binding directly to the HAprotein and inhibiting its normal activity.

In an alternative embodiment, homologues of the HA protein can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the HA protein for HA protein agonist orantagonist activity. In one embodiment, a variegated library of HAvariants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof HA variants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential HA sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of HA sequences therein.There are a variety of methods which can be used to produce libraries ofpotential HA homologues from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential HA sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477.

In addition, libraries of fragments of the HA protein coding can be usedto generate a variegated population of HA fragments for screening andsubsequent selection of homologues of an HA protein. In one embodiment,a library of coding sequence fragments can be generated by treating adouble stranded PCR fragment of an HA coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the HA protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of HA homologues. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify HA homologues (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In another embodiment, cell based assays can be exploited to analyze avariegated HA library, using methods well known in the art.

D. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification of C. glutamicumand related organisms; mapping of genomes of organisms related to C.glutamicum; identification and localization of C. glutamicum sequencesof interest; evolutionary studies; determination of HA protein regionsrequired for function; modulation of an HA protein activity; modulationof the metabolism of one or more inorganic compounds; modulation of themodification or degradation of one or more aromatic or aliphaticcompounds; modulation of cell wall synthesis or rearrangements;modulation of enzyme activity or proteolysis; and modulation of cellularproduction of a desired compound, such as a fine chemical.

The HA nucleic acid molecules of the invention have a variety of uses.First, they may be used to identify an organism as being Corynebacteriumglutamicum or a close relative thereof. Also, they may be used toidentify the presence of C. glutamicum or a relative thereof in a mixedpopulation of microorganisms. The invention provides the nucleic acidsequences of a number of C. glutamicum genes; by probing the extractedgenomic DNA of a culture of a unique or mixed population ofmicroorganisms under stringent conditions with a probe spanning a regionof a C. glutamicum gene which is unique to this organism, one canascertain whether this organism is present. Although Corynebacteriumglutamicum itself is nonpathogenic, it is related to pathogenic species,such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is thecausative agent of diphtheria, a rapidly developing, acute, febrileinfection which involves both local and systemic pathology. In thisdisease, a local lesion develops in the upper respiratory tract andinvolves necrotic injury to epithelial cells; the bacilli secrete toxinwhich is disseminated through this lesion to distal susceptible tissuesof the body. Degenerative changes brought about by the inhibition ofprotein synthesis in these tissues, which include heart, muscle,peripheral nerves, adrenals, kidneys, liver and spleen, result in thesystemic pathology of the disease. Diphtheria continues to have highincidence in many parts of the world, including Africa, Asia, EasternEurope and the independent states of the former Soviet Union. An ongoingepidemic of diphtheria in the latter two regions has resulted in atleast 5,000 deaths since 1990.

In one embodiment, the invention provides a method of identifying thepresence or activity of Cornyebacterium diphtheriae in a subject. Thismethod includes detection of one or more of the nucleic acid or aminoacid sequences of the invention (e.g., the sequences set forth inAppendix A or Appendix B) in a subject, thereby detecting the presenceor activity of Corynebacterium diphtheriae in the subject. C. glutamicumand C. diphtheriae are related bacteria, and many of the nucleic acidand protein molecules in C. glutamicum are homologous to C. diphtheriaenucleic acid and protein molecules, and can therefore be used to detectC. diphtheriae in a subject.

The nucleic acid and protein molecules of the invention may also serveas markers for specific regions of the genome. This has utility not onlyin the mapping of the genome, but also for functional studies of C.glutamicum proteins. For example, to identify the region of the genometo which a particular C. glutamicum DNA-binding protein binds, the C.glutamicum genome could be digested, and the fragments incubated withthe DNA-binding protein. Those which bind the protein may beadditionally probed with the nucleic acid molecules of the invention,preferably with readily detectable labels; binding of such a nucleicacid molecule to the genome fragment enables the localization of thefragment to the genome map of C. glutamicum, and, when performedmultiple times with different enzymes, facilitates a rapid determinationof the nucleic acid sequence to which the protein binds. Further, thenucleic acid molecules of the invention may be sufficiently homologousto the sequences of related species such that these nucleic acidmolecules may serve as markers for the construction of a genomic map inrelated bacteria, such as Brevibacterium lactofermentum.

The HA nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The processes involved inadaptation and the maintenance of homeostasis in which the molecules ofthe invention participate are utilized by a wide variety of species; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein which are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

Manipulation of the HA nucleic acid molecules of the invention mayresult in the production of HA proteins having functional differencesfrom the wild-type HA proteins. These proteins may be improved inefficiency or activity, may be present in greater numbers in the cellthan is usual, or may be decreased in efficiency or activity.

The invention provides methods for screening molecules which modulatethe activity of an HA protein, either by interacting with the proteinitself or a substrate or binding partner of the HA protein, or bymodulating the transcription or translation of an HA nucleic acidmolecule of the invention. In such methods, a microorganism expressingone or more HA proteins of the invention is contacted with one or moretest compounds, and the effect of each test compound on the activity orlevel of expression of the HA protein is assessed.

The modulation of activity or number of HA proteins involved in cellwall biosynthesis or rearrangements may impact the production, yield,and/or efficiency of production of one or more fine chemicals from C.glutamicum cells. For example, by altering the activity of theseproteins, it may be possible to modulate the structure or thickness ofthe cell wall. The cell wall serves in large measure as a protectivedevice against osmotic lysis and external sources of injury; bymodifying the cell wall it may be possible to increase the ability of C.glutamicum to withstand the mechanical and shear force stressesencountered by this microorganism during large-scale fermentor culture.Further, each C. glutamicum cell is surrounded by a thick cell wall, andthus, a significant portion of the biomass present in large scaleculture consists of cell wall. By increasing the rate at which the cellwall is synthesized or by activating cell wall synthesis (throughgenetic engineering of the HA cell wall proteins of the invention) itmay be possible to improve the growth rate of the microorganism.Similarly, by decreasing the activity or number of proteins involved inthe degradation of cell wall or by decreasing the repression of cellwall biosynthesis, an overall increase in cell wall production may beachieved. An increase in the number of viable C. glutamicum cells (asmay be accomplished by any of the foregoing described proteinalterations) should result in increased numbers of cells producing thedesired fine chemical in large-scale fermentor culture, which shouldpermit increased yields or efficiency of production of these compoundsfrom the culture.

The modulation of activity or number of C. glutamicum HA proteins thatparticipate in the modification or degradation of aromatic or aliphaticcompounds may also have direct or indirect impacts on the production ofone or more fine chemicals from these cells. Certain aromatic oraliphatic modification or degradation products are desirable finechemicals (e.g., organic acids or modified aromatic and aliphaticcompounds); thus, by modifying the enzymes which perform thesemodifications (e.g., hydroxylation, methylation, or isomerization) ordegradation reactions, it may be possible to increase the yields ofthese desired compounds. Similarly, by decreasing the activity or numberof proteins involved in pathways which further degrade the modified orbreakdown products of the aforementioned reactions it may be possible toimprove the yields of these fine chemicals from C. glutamicum cells inculture.

These aromatic and aliphatic modification and degradative enzymes arethemselves fine chemicals. In purified form, these enzymes may be usedto degrade aromatic and aliphatic compounds (e.g., toxic chemicals suchas petroleum products), either for the bioremediation of polluted sites,for the engineered decomposition of wastes, or for the large-scale andeconomically feasible production of desired modified aromatic oraliphatic compounds or their breakdown products, some of which may beconveniently used as carbon or energy sources for other finechemical-producing compounds in culture (see, e.g., Faber, K. (1995)Biotransformations in Organic Chemistry, Springer: Berlin and referencestherein; and Roberts, S. M., ed. (1992-1996) PreparativeBiotransformations, Wiley: Chichester, and references therein). Bygenetically altering these proteins such that their regulation by othercellular mechanisms is lessened or abolished, it may be possible toincrease the overall number or activity of these proteins, therebyimproving not only the yield of these fine chemicals but also theactivity of these harvested proteins.

The modification of these aromatic and aliphatic modifying anddegradation enzymes may also have an indirect effect on the productionof one or more fine chemical. Many aromatic and aliphatic compounds(such as those that may be encountered as impurities in culture media oras waste products from cellular metabolism) are toxic to cells; bymodifying and/or degrading these compounds such that they may be readilyremoved or destroyed, cellular viability should be increased. Further,these enzymes may modify or degrade these compounds in such a mannerthat the resulting products may enter the normal carbon metabolismpathways of the cell, thus rendering the cell able to use thesecompounds as alternate carbon or energy sources. In large-scale culturesituations, when there may be limiting amounts of optimal carbonsources, these enzymes provide a method by which cells may continue togrow and divide using aromatic or aliphatic compounds as nutrients. Ineither case, the resulting increase in the number of C. glutamicum cellsin the culture producing the desired fine chemical should in turn resultin increased yields or efficiency of production of the fine chemical(s).

Modifications in activity or number of HA proteins involved in themetabolism of inorganic compounds may also directly or indirectly affectthe production of one or more fine chemicals from C. glutamicum orrelated bacterial cultures. For example, many desirable fine chemicals,such as nucleic acids, amino acids, cofactors and vitamins (e.g.,thiamine, biotin, and lipoic acid) cannot be synthesized withoutinorganic molecules such as phosphorous, nitrate, sulfate, and iron. Theinorganic metabolism proteins of the invention permit the cell to obtainthese molecules from a variety of inorganic compounds and to divert theminto various fine chemical biosynthetic pathways. Therefore, byincreasing the activity or number of enzymes involved in the metabolismof these inorganic compounds, it may be possible to increase the supplyof these possibly limiting inorganic molecules, thereby directlyincreasing the production or efficiency of production of various finechemicals from C. glutamicum cells containing such altered proteins.Modification of the activity or number of inorganic metabolism enzymesof the invention may also render C. glutamicum able to better utilizelimited inorganic compound supplies, or to utilize nonoptimal inorganiccompounds to synthesize amino acids, vitamins, cofactors, or nucleicacids, all of which are necessary for continued growth and replicationof the cell. By improving the viability of these cells in large-scaleculture, the number of C. glutamicum cells producing one or more finechemicals in the culture may also be increased, in turn increasing theyields or efficiency of production of one or more fine chemicals.

C. glutamicum enzymes for general processes are themselves desirablefine chemicals. The specific properties of enzymes (i.e., regio- andstereospecificity, among others) make them useful catalysts for chemicalreactions in vitro. Either whole C. glutamicum cells may be incubatedwith an appropriate substrate such that the desired product is producedby enzymes in the cell, or the desired enzymes may be overproduced andpurified from C. glutamicum cultures (or those of a related bacterium)and subsequently utilized in in vitro reactions in an industrial setting(either in solution or immobilized on a suitable immobile phase). Ineither situation, the enzyme can either be a natural C. glutamicumprotein, or it may be mutagenized to have an altered activity; typicalindustrial uses for such enzymes include as catalysts in the chemicalindustry (e.g., for synthetic organic chemistry) as food additives, asfeed components, for fruit processing, for leather preparation, indetergents, in analysis and medicine, and in the textile industry (see,e.g., Yamada, H. (1993) “Microbial reactions for the production ofuseful organic compounds,” Chimica 47: 5-10; Roberts, S. M. (1998)Preparative biotransformations: the employment of enzymes andwhole-cells in synthetic chemistry,” J. Chem. Soc. Perkin Trans. 1:157-169; Zaks, A. and Dodds, D. R. (1997) “Application of biocatalysisand biotransformations to the synthesis of pharmaceuticals,” DDT2:513-531; Roberts, S. M. and Williamson, N. M. (1997) “The use of enzymesfor the preparation of biologically active natural products andanalogues in optically active form,” Curr. Organ. Chemistry 1:1-20;Faber, K. (1995) Biotransformations in Organic Chemistry, Springer:Berlin; Roberts, S. M., ed. (1992-96) Preparative Biotransformations,Wiley: Chichester; Cheetham, P. S. J. (1995) “The applications ofenzymes in industry” in: Handbook of Enzyme Biotechnology, 3^(rd) ed.,Wiseman, A., ed., Elis: Horwood, p. 419-552; and Ullmann's Encyclopediaof Industrial Chemistry (1987), vol. A9, Enzymes, p. 390-457). Thus, byincreasing the activity or number of these enzymes, it may be possibleto also increase the ability of the cell to convert supplied substratesto desired products, or to overproduce these enzymes for increasedyields in large-scale culture. Further, by mutagenizing these proteinsit may be possible to remove feedback inhibition or other repressivecellular regulatory controls such that greater numbers of these enzymesmay be produced and activated by the cell, thereby leading to greateryields, production, or efficiency of production of these fine chemicalproteins from large-scale cultures. Further, manipulation of theseenzymes may alter the activity of one or more C. glutamicum metabolicpathways, such as those for the biosynthesis or secretion of one or morefine chemicals.

Mutagenesis of the proteolytic enzymes of the invention such that theyare altered in activity or number may also directly or indirectly affectthe yield, production, and/or efficiency of production of one or morefine chemicals from C. glutamicum. For example, by increasing theactivity or number of these proteins, it may be possible to increase theability of the bacterium to survive in large-scale culture, due to anincreased ability of the cell to rapidly degrade proteins misfolded inresponse to the high temperatures, nonoptimal pH, and other stressesencountered during fermentor culture. Increased numbers of cells inthese cultures may result in increased yields or efficiency ofproduction of one or more desired fine chemicals, due to the relativelylarger number of cells producing these compounds in the culture. Also,C. glutamicum cells possess multiple cell-surface proteases which serveto break down external nutrients into molecules which may be morereadily incorporated by the cells as carbon/energy sources or nutrientsof other kinds. An increase in activity or number of these enzymes mayimprove this turnover and increase the levels of available nutrients,thereby improving cell growth or production. Thus, modifications of theproteases of the invention may indirectly impact C. glutamicum finechemical production.

A more direct impact on fine chemical production in response to themodification of one or more of the proteases of the invention may occurwhen these proteases are involved in the production or degradation of adesired fine chemical. By decreasing the activity of a protease whichdegrades a fine chemical or a protein involved in the synthesis of afine chemical it may be possible to increase the levels of that finechemical (due to the decreased degradation or increased synthesis of thecompound). Similarly, by increasing the activity of a protease whichdegrades a compound to result in a fine chemical or a protein involvedin the degradation of a fine chemical, a similar result should beachieved: increased levels of the desired fine chemical from C.glutamicum cells containing these engineered proteins.

The aforementioned mutagenesis strategies for HA proteins to result inincreased yields of a fine chemical from C. glutamicum are not meant tobe limiting; variations on these strategies will be readily apparent toone of ordinary skill in the art. Using such strategies, andincorporating the mechanisms disclosed herein, the nucleic acid andprotein molecules of the invention may be utilized to generate C.glutamicum or related strains of bacteria expressing mutated HA nucleicacid and protein molecules such that the yield, production, and/orefficiency of production of a desired compound is improved. This desiredcompound may be any product produced by C. glutamicum, which includesthe final products of biosynthesis pathways and intermediates ofnaturally-occurring metabolic pathways, as well as molecules which donot naturally occur in the metabolism of C. glutamicum, but which areproduced by a C. glutamicum strain of the invention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patent applications, patents, published patent applications, Tables,Appendices, and the sequence listing cited throughout this applicationare hereby incorporated by reference.

EXEMPLIFICATION Example 1 Preparation of Total Genomic DNA ofCorynebacterium glutamicum ATCC 13032

A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnightat 30° C. with vigorous shaking in BHI medium (Difco). The cells wereharvested by centrifugation, the supernatant was discarded and the cellswere resuspended in 5 ml buffer-I (5% of the original volume of theculture—all indicated volumes have been calculated for 100 ml of culturevolume). Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/lMgSO₄x7H₂O, 10 ml/l KH₂PO₄ solution (100 g/l, adjusted to pH 6.7 withKOH), 50 ml/l M12 concentrate (10 g/l (NH₄)₂SO₄, 1 g/l NaCl, 2 g/lMgSO₄x7H₂O, 0.2 g/l CaCl₂, 0.5 g/l yeast extract (Difco), 10 ml/ltrace-elements-mix (200 mg/l FeSO₄xH₂O, 10 mg/l ZnSO₄x7H₂O, 3 mg/lMnCl₂x4H₂O, 30 mg/l H₃BO₃ 20 mg/l CoCl₂x6H₂O, 1 mg/l NiCl₂x6H₂O, 3 mg/lNa₂MoO₄x2H₂O, 500 mg/l complexing agent (EDTA or critic acid), 100 ml/lvitamins-mix (0.2 mg/l biotin, 0.2 mg/l folic acid, 20 mg/l p-aminobenzoic acid, 20 mg/l riboflavin, 40 mg/l ca-panthothenate, 140 mg/lnicotinic acid, 40 mg/l pyridoxole hydrochloride, 200 mg/lmyo-inositol). Lysozyme was added to the suspension to a finalconcentration of 2.5 mg/ml. After an approximately 4 h incubation at 37°C., the cell wall was degraded and the resulting protoplasts areharvested by centrifugation. The pellet was washed once with 5 mlbuffer-I and once with 5 ml TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8).The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution(10%) and 0.5 ml NaCl solution (5 M) are added. After adding ofproteinase K to a final concentration of 200 μg/ml, the suspension isincubated for ca. 18 h at 37° C. The DNA was purified by extraction withphenol, phenol-chloroform-isoamylalcohol and chloroform-isoamylalcoholusing standard procedures. Then, the DNA was precipitated by adding 1/50volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30min incubation at −20° C. and a 30 min centrifugation at 12,000 rpm in ahigh speed centrifuge using a SS34 rotor (Sorvall). The DNA wasdissolved in 1 ml TE-buffer containing 20 μg/ml RNaseA and dialysed at4° C. against 1000 ml TE-buffer for at least 3 hours. During this time,the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysedDNA solution, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol are added. Aftera 30 min incubation at −20° C., the DNA was collected by centrifugation(13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pelletwas dissolved in TE-buffer. DNA prepared by this procedure could be usedfor all purposes, including southern blotting or construction of genomiclibraries.

Example 2 Construction of Genomic Libraries in Escherichia coli ofCorynebacterium glutamicum ATCC13032

Using DNA prepared as described in Example 1, cosmid and plasmidlibraries were constructed according to known and well establishedmethods (see e.g., Sambrook, J. et al. (1989) “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons.)

Any plasmid or cosmid could be used. Of particular use were the plasmidspBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci. USA,75:3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK− and others;Stratagene, LaJolla, USA), or cosmids as SuperCos1 (Stratagene, LaJolla,USA) or Lorist6 (Gibson, T. J., Rosenthal A. and Waterson, R. H. (1987)Gene 53:283-286. Gene libraries specifically for use in C. glutamicummay be constructed using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey(1994) J. Microbiol. Biotechnol. 4: 256-263).

Example 3 DNA Sequencing and Computational Functional Analysis

Genomic libraries as described in Example 2 were used for DNA sequencingaccording to standard methods, in particular by the chain terminationmethod using ABI377 sequencing machines (see e.g., Fleischman, R. D. etal. (1995) “Whole-genome Random Sequencing and Assembly of HaemophilusInfluenzae Rd., Science, 269:496-512). Sequencing primers with thefollowing nucleotide sequences were used: 5′-GGAAACAGTATGACCATG-3′ or5′-GTAAAACGACGGCCAGT-3′.

Example 4 In Vivo Mutagenesis

In vivo mutagenesis of Corynebacterium glutamicum can be performed bypassage of plasmid (or other vector) DNA through E. coli or othermicroorganisms (e.g. Bacillus spp. or yeasts such as Saccharomycescerevisiae) which are impaired in their capabilities to maintain theintegrity of their genetic information. Typical mutator strains havemutations in the genes for the DNA repair system (e.g., mutHLS, mutD,mutT, etc.; for reference, see Rupp, W. D. (1996) DNA repair mechanisms,in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.)Such strains are well known to those of ordinary skill in the art. Theuse of such strains is illustrated, for example, in Greener, A. andCallahan, M. (1994) Strategies 7: 32-34.

Example 5 DNA Transfer Between Escherichia coli and Corynebacteriumglutamicum

Several Corynebacterium and Brevibacterium species contain endogenousplasmids (as e.g., pHM1519 or pBL1) which replicate autonomously (forreview see, e.g., Martin, J. F. et al. (1987) Biotechnology, 5:137-146).Shuttle vectors for Escherichia coli and Corynebacterium glutamicum canbe readily constructed by using standard vectors for E. coli (Sambrook,J. et al. (1989), “Molecular Cloning: A Laboratory Manual”, Cold SpringHarbor Laboratory Press or Ausubel, F. M. et al. (1994) “CurrentProtocols in Molecular Biology”, John Wiley & Sons) to which a origin orreplication for and a suitable marker from Corynebacterium glutamicum isadded. Such origins of replication are preferably taken from endogenousplasmids isolated from Corynebacterium and Brevibacterium species. Ofparticular use as transformation markers for these species are genes forkanamycin resistance (such as those derived from the Tn5 or Tn903transposons) or chloramphenicol (Winnacker, E. L. (1987) “From Genes toClones—Introduction to Gene Technology, VCH, Weinheim). There arenumerous examples in the literature of the construction of a widevariety of shuttle vectors which replicate in both E. coli and C.glutamicum, and which can be used for several purposes, including geneover-expression (for reference, see e.g., Yoshihama, M. et al. (1985) J.Bacteriol. 162:591-597, Martin J. F. et al. (1987) Biotechnology,5:137-146 and Eikmanns, B. J. et al. (1991) Gene, 102:93-98).

Using standard methods, it is possible to clone a gene of interest intoone of the shuttle vectors described above and to introduce such ahybrid vectors into strains of Corynebacterium glutamicum.Transformation of C. glutamicum can be achieved by protoplasttransformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311),electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters,53:399-303) and in cases where special vectors are used, also byconjugation (as described e.g. in Schafer, A et al. (1990) J. Bacteriol.172:1663-1666). It is also possible to transfer the shuttle vectors forC. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum(using standard methods well-known in the art) and transforming it intoE. coli. This transformation step can be performed using standardmethods, but it is advantageous to use an Mcr-deficient E. coli strain,such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).

Genes may be overexpressed in C. glutamicum strains using plasmids whichcomprise pCG1 (U.S. Pat. No. 4,617,267) or fragments thereof, andoptionally the gene for kanamycin resistance from TN903 (Grindley, N. D.and Joyce, C. M. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180).In addition, genes may be overexpressed in C. glutamicum strains usingplasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J. Microbiol.Biotechnol. 4: 256-263).

Aside from the use of replicative plasmids, gene overexpression can alsobe achieved by integration into the genome. Genomic integration in C.glutamicum or other Corynebacterium or Brevibacterium species may beaccomplished by well-known methods, such as homologous recombinationwith genomic region(s), restriction endonuclease mediated integration(REMI) (see, e.g., DE Patent 19823834), or through the use oftransposons. It is also possible to modulate the activity of a gene ofinterest by modifying the regulatory regions (e.g., a promoter, arepressor, and/or an enhancer) by sequence modification, insertion, ordeletion using site-directed methods (such as homologous recombination)or methods based on random events (such as transposon mutagenesis orREMI). Nucleic acid sequences which function as transcriptionalterminators may also be inserted 3′ to the coding region of one or moregenes of the invention; such terminators are well-known in the art andare described, for example, in Winnacker, E. L. (1987) From Genes toClones—Introduction to Gene Technology. VCH: Weinheim.

Example 6 Assessment of the Expression of the Mutant Protein

Observations of the activity of a mutated protein in a transformed hostcell rely on the fact that the mutant protein is expressed in a similarfashion and in a similar quantity to that of the wild-type protein. Auseful method to ascertain the level of transcription of the mutant gene(an indicator of the amount of mRNA available for translation to thegene product) is to perform a Northern blot (for reference see, forexample, Ausubel et al. (1988) Current Protocols in Molecular Biology,Wiley: New York), in which a primer designed to bind to the gene ofinterest is labeled with a detectable tag (usually radioactive orchemiluminescent), such that when the total RNA of a culture of theorganism is extracted, run on gel, transferred to a stable matrix andincubated with this probe, the binding and quantity of binding of theprobe indicates the presence and also the quantity of mRNA for thisgene. This information is evidence of the degree of transcription of themutant gene. Total cellular RNA can be prepared from Corynebacteriumglutamicum by several methods, all well-known in the art, such as thatdescribed in Bormann, E. R. et al. (1992) Mol. Microbiol. 6: 317-326.

To assess the presence or relative quantity of protein translated fromthis mRNA, standard techniques, such as a Western blot, may be employed(see, for example, Ausubel et al. (1988) Current Protocols in MolecularBiology, Wiley: New York). In this process, total cellular proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose, and incubated with a probe, such as an antibody,which specifically binds to the desired protein. This probe is generallytagged with a chemiluminescent or colorimetric label which may bereadily detected. The presence and quantity of label observed indicatesthe presence and quantity of the desired mutant protein present in thecell.

Example 7 Growth of Genetically Modified Corynebacteriumglutamicum—Media and Culture Conditions

Genetically modified Corynebacteria are cultured in synthetic or naturalgrowth media. A number of different growth media for Corynebacteria areboth well-known and readily available (Lieb et al. (1989) Appl.Microbiol. Biotechnol., 32:205-210; von der Osten et al. (1998)Biotechnology Letters, 11:11-16; Patent DE 4,120,867; Liebl (1992) “TheGenus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. etal., eds. Springer-Verlag). These media consist of one or more carbonsources, nitrogen sources, inorganic salts, vitamins and trace elements.Preferred carbon sources are sugars, such as mono-, di-, orpolysaccharides. For example, glucose, fructose, mannose, galactose,ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starchor cellulose serve as very good carbon sources. It is also possible tosupply sugar to the media via complex compounds such as molasses orother by-products from sugar refinement. It can also be advantageous tosupply mixtures of different carbon sources. Other possible carbonsources are alcohols and organic acids, such as methanol, ethanol,acetic acid or lactic acid. Nitrogen sources are usually organic orinorganic nitrogen compounds, or materials which contain thesecompounds. Exemplary nitrogen sources include ammonia gas or ammoniasalts, such as NH₄Cl or (NH₄)₂SO₄, NH₄OH, nitrates, urea, amino acids orcomplex nitrogen sources like corn steep liquor, soy bean flour, soybean protein, yeast extract, meat extract and others.

Inorganic salt compounds which may be included in the media include thechloride-, phosphorous- or sulfate-salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Chelating compounds can be added to the medium to keep the metal ions insolution. Particularly useful chelating compounds includedihydroxyphenols, like catechol or protocatechuate, or organic acids,such as citric acid. It is typical for the media to also contain othergrowth factors, such as vitamins or growth promoters, examples of whichinclude biotin, riboflavin, thiamin, folic acid, nicotinic acid,pantothenate and pyridoxin. Growth factors and salts frequentlyoriginate from complex media components such as yeast extract, molasses,corn steep liquor and others. The exact composition of the mediacompounds depends strongly on the immediate experiment and isindividually decided for each specific case. Information about mediaoptimization is available in the textbook “Applied Microbiol.Physiology, A Practical Approach (eds. P. M. Rhodes, P. F. Stanbury, IRLPress (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible toselect growth media from commercial suppliers, like standard 1 (Merck)or BHI (grain heart infusion, DIFCO) or others.

All medium components are sterilized, either by heat (20 minutes at 1.5bar and 121° C.) or by sterile filtration. The components can either besterilized together or, if necessary, separately. All media componentscan be present at the beginning of growth, or they can optionally beadded continuously or batchwise.

Culture conditions are defined separately for each experiment. Thetemperature should be in a range between 15° C. and 45° C. Thetemperature can be kept constant or can be altered during theexperiment. The pH of the medium should be in the range of 5 to 8.5,preferably around 7.0, and can be maintained by the addition of buffersto the media. An exemplary buffer for this purpose is a potassiumphosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and otherscan alternatively or simultaneously be used. It is also possible tomaintain a constant culture pH through the addition of NaOH or NH₄OHduring growth. If complex medium components such as yeast extract areutilized, the necessity for additional buffers may be reduced, due tothe fact that many complex compounds have high buffer capacities. If afermentor is utilized for culturing the micro-organisms, the pH can alsobe controlled using gaseous ammonia.

The incubation time is usually in a range from several hours to severaldays. This time is selected in order to permit the maximal amount ofproduct to accumulate in the broth. The disclosed growth experiments canbe carried out in a variety of vessels, such as microtiter plates, glasstubes, glass flasks or glass or metal fermentors of different sizes. Forscreening a large number of clones, the microorganisms should becultured in microtiter plates, glass tubes or shake flasks, either withor without baffles. Preferably 100 ml shake flasks are used, filled with10% (by volume) of the required growth medium. The flasks should beshaken on a rotary shaker (amplitude 25 mm) using a speed-range of100-300 rpm. Evaporation losses can be diminished by the maintenance ofa humid atmosphere; alternatively, a mathematical correction forevaporation losses should be performed.

If genetically modified clones are tested, an unmodified control cloneor a control clone containing the basic plasmid without any insertshould also be tested. The medium is inoculated to an OD₆₀₀ of 0.5-1.5using cells grown on agar plates, such as CM plates (10 g/l glucose, 2,5g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/lmeat extract, 22 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeastextract, 5 g/l meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that hadbeen incubated at 30° C. Inoculation of the media is accomplished byeither introduction of a saline suspension of C. glutamicum cells fromCM plates or addition of a liquid preculture of this bacterium.

Example 8 In Vitro Analysis of the Function of Mutant Proteins

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one ofordinary skill in the art. Overviews about enzymes in general, as wellas specific details concerning structure, kinetics, principles, methods,applications and examples for the determination of many enzymeactivities may be found, for example, in the following references:Dixon, M., and Webb, E. C., (1979) Enzymes. Longmans: London; Fersht,(1985) Enzyme Structure and Mechanism. Freeman: New York; Walsh, (1979)Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N. C.,Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press:Oxford; Boyer, P. D., ed. (1983) The Enzymes, 3^(rd) ed. Academic Press:New York; Bisswanger, H., (1994) Enzymkinetik, 2^(nd) ed. VCH: Weinheim(ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβ1, M., eds.(1983-1986) Methods of Enzymatic Analysis, 3^(rd) ed., vol. I-XII,Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of IndustrialChemistry (1987) vol. A9, “Enzymes”. VCH: Weinheim, p. 352-363.

The activity of proteins which bind to DNA can be measured by severalwell-established methods, such as DNA band-shift assays (also called gelretardation assays). The effect of such proteins on the expression ofother molecules can be measured using reporter gene assays (such as thatdescribed in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 andreferences cited therein). Reporter gene test systems are well known andestablished for applications in both pro- and eukaryotic cells, usingenzymes such as beta-galactosidase, green fluorescent protein, andseveral others.

The determination of activity of membrane-transport proteins can beperformed according to techniques such as those described in Gennis, R.B. (1989) “Pores, Channels and Transporters”, in Biomembranes, MolecularStructure and Function, Springer: Heidelberg, p. 85-137; 199-234; and270-322.

Example 9 Analysis of Impact of Mutant Protein on the Production of theDesired Product

The effect of the genetic modification in C. glutamicum on production ofa desired compound (such as an amino acid) can be assessed by growingthe modified microorganism under suitable conditions (such as thosedescribed above) and analyzing the medium and/or the cellular componentfor increased production of the desired product (i.e., an amino acid).Such analysis techniques are well known to one of ordinary skill in theart, and include spectroscopy, thin layer chromatography, stainingmethods of various kinds, enzymatic and microbiological methods, andanalytical chromatography such as high performance liquid chromatography(see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol.A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al.,(1987) “Applications of HPLC in Biochemistry” in: Laboratory Techniquesin Biochemistry and Molecular Biology, vol. 17; Rehm et al. (1993)Biotechnology, vol. 3, Chapter III: “Product recovery and purification”,page 469-714, VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations:downstream processing for biotechnology, John Wiley and Sons; Kennedy,J. F. and Cabral, J. M. S. (1992) Recovery processes for biologicalmaterials, John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988)Biochemical separations, in: Ulmann's Encyclopedia of IndustrialChemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications.)

In addition to the measurement of the final product of fermentation, itis also possible to analyze other components of the metabolic pathwaysutilized for the production of the desired compound, such asintermediates and side-products, to determine the overall efficiency ofproduction of the compound. Analysis methods include measurements ofnutrient levels in the medium (e.g., sugars, hydrocarbons, nitrogensources, phosphate, and other ions), measurements of biomass compositionand growth, analysis of the production of common metabolites ofbiosynthetic pathways, and measurement of gasses produced duringfermentation. Standard methods for these measurements are outlined inApplied Microbial Physiology, A Practical Approach, P. M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN:0199635773) and references cited therein.

Example 10 Purification of the Desired Product from C. glutamicumCulture

Recovery of the desired product from the C. glutamicum cells orsupernatant of the above-described culture can be performed by variousmethods well known in the art. If the desired product is not secretedfrom the cells, the cells can be harvested from the culture by low-speedcentrifugation, the cells can be lysed by standard techniques, such asmechanical force or sonication. The cellular debris is removed bycentrifugation, and the supernatant fraction containing the solubleproteins is retained for further purification of the desired compound.If the product is secreted from the C. glutamicum cells, then the cellsare removed from the culture by low-speed centrifugation, and thesupernate fraction is retained for further purification.

The supernatant fraction from either purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on a chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by the resinwhile the sample is not. Such chromatography steps may be repeated asnecessary, using the same or different chromatography resins. One ofordinary skill in the art would be well-versed in the selection ofappropriate chromatography resins and in their most efficaciousapplication for a particular molecule to be purified. The purifiedproduct may be concentrated by filtration or ultrafiltration, and storedat a temperature at which the stability of the product is maximized.

There are a wide array of purification methods known to the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey, J. E. &Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: New York(1986).

The identity and purity of the isolated compounds may be assessed bytechniques standard in the art. These include high-performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, NIRS, enzymatic assay, or microbiologically. Suchanalysis methods are reviewed in: Patek et al. (1994) Appl. Environ.Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11:27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70.Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH:Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p.581-587; Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, vol. 17.

Example 11 Analysis of the Gene Sequences of the Invention

The comparison of sequences and determination of percent homologybetween two sequences are art-known techniques, and can be accomplishedusing a mathematical algorithm, such as the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Suchan algorithm is incorporated into the NBLAST and XBLAST programs(version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to HA nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to HA protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one ofordinary skill in the art will know how to optimize the parameters ofthe program (e.g., XBLAST and NBLAST) for the specific sequence beinganalyzed.

Another example of a mathematical algorithm utilized for the comparisonof sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl.Biosci. 4: 11-17). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art, and include ADVANCE and ADAM.described in Torelli and Robotti (1994) Comput. Appl. Biosci. 10:3-5;and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.

The percent homology between two amino acid sequences can also beaccomplished using the GAP program in the GCG software package(available at http://www.gcg.com), using either a Blosum 62 matrix or aPAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a lengthweight of 2, 3, or 4. The percent homology between two nucleic acidsequences can be accomplished using the GAP program in the GCG softwarepackage, using standard parameters, such as a gap weight of 50 and alength weight of 3.

A comparative analysis of the gene sequences of the invention with thosepresent in Genbank has been performed using techniques known in the art(see, e.g., Bexevanis and Ouellette, eds. (1998) Bioinformatics: APractical Guide to the Analysis of Genes and Proteins. John Wiley andSons: New York). The gene sequences of the invention were compared togenes present in Genbank in a three-step process. In a first step, aBLASTN analysis (e.g., a local alignment analysis) was performed foreach of the sequences of the invention against the nucleotide sequencespresent in Genbank, and the top 500 hits were retained for furtheranalysis. A subsequent FASTA search (e.g., a combined local and globalalignment analysis, in which limited regions of the sequences arealigned) was performed on these 500 hits. Each gene sequence of theinvention was subsequently globally aligned to each of the top threeFASTA hits, using the GAP program in the GCG software package (usingstandard parameters). In order to obtain correct results, the length ofthe sequences extracted from Genbank were adjusted to the length of thequery sequences by methods well-known in the art. The results of thisanalysis are set forth in Table 4. The resulting data is identical tothat which would have been obtained had a GAP (global) analysis alonebeen performed on each of the genes of the invention in comparison witheach of the references in Genbank, but required significantly reducedcomputational time as compared to such a database-wide GAP (global)analysis. Sequences of the invention for which no alignments above thecutoff values were obtained are indicated on Table 4 by the absence ofalignment information. It will further be understood by one of ordinaryskill in the art that the GAP alignment homology percentages set forthin Table 4 under the heading “% homology (GAP)” are listed in theEuropean numerical format, wherein a ‘,’ represents a decimal point. Forexample, a value of “40,345” in this column represents “40.345%”.

Example 12 Construction and Operation of DNA Microarrays

The sequences of the invention may additionally be used in theconstruction and application of DNA microarrays (the design,methodology, and uses of DNA arrays are well known in the art, and aredescribed, for example, in Schena, M. et al. (1995) Science 270:467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367;DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi,J. L. et al. (1997) Science 278: 680-686).

DNA microarrays are solid or flexible supports consisting ofnitrocellulose, nylon, glass, silicone, or other materials. Nucleic acidmolecules may be attached to the surface in an ordered manner. Afterappropriate labeling, other nucleic acids or nucleic acid mixtures canbe hybridized to the immobilized nucleic acid molecules, and the labelmay be used to monitor and measure the individual signal intensities ofthe hybridized molecules at defined regions. This methodology allows thesimultaneous quantification of the relative or absolute amount of all orselected nucleic acids in the applied nucleic acid sample or mixture.DNA microarrays, therefore, permit an analysis of the expression ofmultiple (as many as 6800 or more) nucleic acids in parallel (see, e.g.,Schena, M. (1996) BioEssays 18(5): 427-431).

The sequences of the invention may be used to design oligonucleotideprimers which are able to amplify defined regions of one or more C.glutamicum genes by a nucleic acid amplification reaction such as thepolymerase chain reaction. The choice and design of the 5′ or 3′oligonucleotide primers or of appropriate linkers allows the covalentattachment of the resulting PCR products to the surface of a supportmedium described above (and also described, for example, Schena, M. etal. (1995) Science 270: 467-470).

Nucleic acid microarrays may also be constructed by in situoligonucleotide synthesis as described by Wodicka, L. et al. (1997)Nature Biotechnology 15: 1359-1367. By photolithographic methods,precisely defined regions of the matrix are exposed to light. Protectivegroups which are photolabile are thereby activated and undergonucleotide addition, whereas regions that are masked from light do notundergo any modification. Subsequent cycles of protection and lightactivation permit the synthesis of different oligonucleotides at definedpositions. Small, defined regions of the genes of the invention may besynthesized on microarrays by solid phase oligonucleotide synthesis.

The nucleic acid molecules of the invention present in a sample ormixture of nucleotides may be hybridized to the microarrays. Thesenucleic acid molecules can be labeled according to standard methods. Inbrief, nucleic acid molecules (e.g., mRNA molecules or DNA molecules)are labeled by the incorporation of isotopically or fluorescentlylabeled nucleotides, e.g., during reverse transcription or DNAsynthesis. Hybridization of labeled nucleic acids to microarrays isdescribed (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al.(1997), supra; and DeSaizieu A. et al. (1998), supra). The detection andquantification of the hybridized molecule are tailored to the specificincorporated label. Radioactive labels can be detected, for example, asdescribed in Schena, M. et al. (1995) supra) and fluorescent labels maybe detected, for example, by the method of Shalon et al. (1996) GenomeResearch 6: 639-645).

The application of the sequences of the invention to DNA microarraytechnology, as described above, permits comparative analyses ofdifferent strains of C. glutamicum or other Corynebacteria. For example,studies of inter-strain variations based on individual transcriptprofiles and the identification of genes that are important for specificand/or desired strain properties such as pathogenicity, productivity andstress tolerance are facilitated by nucleic acid array methodologies.Also, comparisons of the profile of expression of genes of the inventionduring the course of a fermentation reaction are possible using nucleicacid array technology.

Example 13 Analysis of the Dynamics of Cellular Protein Populations(Proteomics)

The genes, compositions, and methods of the invention may be applied tostudy the interactions and dynamics of populations of proteins, termed‘proteomics’. Protein populations of interest include, but are notlimited to, the total protein population of C. glutamicum (e.g., incomparison with the protein populations of other organisms), thoseproteins which are active under specific environmental or metabolicconditions (e.g., during fermentation, at high or low temperature, or athigh or low pH), or those proteins which are active during specificphases of growth and development.

Protein populations can be analyzed by various well-known techniques,such as gel electrophoresis. Cellular proteins may be obtained, forexample, by lysis or extraction, and may be separated from one anotherusing a variety of electrophoretic techniques. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) separates proteins largelyon the basis of their molecular weight. Isoelectric focusingpolyacrylamide gel electrophoresis (IEF-PAGE) separates proteins bytheir isoelectric point (which reflects not only the amino acid sequencebut also posttranslational modifications of the protein). Another, morepreferred method of protein analysis is the consecutive combination ofboth IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described,for example, in Hermann et al. (1998) Electrophoresis 19: 3217-3221;Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al.(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997)Electrophoresis 18: 1451-1463). Other separation techniques may also beutilized for protein separation, such as capillary gel electrophoresis;such techniques are well known in the art.

Proteins separated by these methodologies can be visualized by standardtechniques, such as by staining or labeling. Suitable stains are knownin the art, and include Coomassie Brilliant Blue, silver stain, orfluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion ofradioactively labeled amino acids or other protein precursors (e.g.,³⁵S-methionine, ³⁵S-cysteine, ¹⁴C-labelled amino acids, ¹⁵N-amino acids,¹⁵NO₃ or ¹⁵NH₄ ⁺ or ¹³C-labelled amino acids) in the medium of C.glutamicum permits the labeling of proteins from these cells prior totheir separation. Similarly, fluorescent labels may be employed. Theselabeled proteins can be extracted, isolated and separated according tothe previously described techniques.

Proteins visualized by these techniques can be further analyzed bymeasuring the amount of dye or label used. The amount of a given proteincan be determined quantitatively using, for example, optical methods andcan be compared to the amount of other proteins in the same gel or inother gels. Comparisons of proteins on gels can be made, for example, byoptical comparison, by spectroscopy, by image scanning and analysis ofgels, or through the use of photographic films and screens. Suchtechniques are well-known in the art.

To determine the identity of any given protein, direct sequencing orother standard techniques may be employed. For example, N- and/orC-terminal amino acid sequencing (such as Edman degradation) may beused, as may mass spectrometry (in particular MALDI or ESI techniques(see, e.g., Langen et al. (1997) Electrophoresis 18: 1184-1192)). Theprotein sequences provided herein can be used for the identification ofC. glutamicum proteins by these techniques.

The information obtained by these methods can be used to comparepatterns of protein presence, activity, or modification betweendifferent samples from various biological conditions (e.g., differentorganisms, time points of fermentation, media conditions, or differentbiotopes, among others). Data obtained from such experiments alone, orin combination with other techniques, can be used for variousapplications, such as to compare the behavior of various organisms in agiven (e.g., metabolic) situation, to increase the productivity ofstrains which produce fine chemicals or to increase the efficiency ofthe production of fine chemicals.

EQUIVALENTS

Those of ordinary skill in the art will recognize, or will be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.TABLE 1 Genes in the Application Nucleic Amino Acid Acid SEQ SEQIdentification ID NO ID NO Code Contig. NT Start NT Stop Function 1 2RXA02548 GR00727 3 293 SULFATE ADENYLATE TRANSFERASE SUBUNIT 2 (EC2.7.7.4) 3 4 RXN00249 VV0057 36825 35869 ADENYLYLSULFATE KINASE (EC2.7.1.25) 5 6 F RXA00249 GR00037 8837 7884 ADENYLYLSULFATE KINASE (EC2.7.1.25) 7 8 RXA01073 GR00300 1274 2104 NH(3)-DEPENDENT NAD(+)SYNTHETASE (EC 6.3.5.1) Urease 9 10 RXN02913 VV0020 8998 8513 UREASEBETA SUBUNIT (EC 3.5.1.5) 11 12 F RXA02264 GR00655 123 4 UREASE ALPHASUBUNIT (EC 3.5.1.5) 13 14 RXN02274 VV0020 8509 6800 UREASE ALPHASUBUNIT (EC 3.5.1.5) 15 16 F RXA02274 GR00656 3 1604 UREASE ALPHASUBUNIT (EC 3.5.1.5) 17 18 RXA02265 GR00655 452 153 UREASE GAMMA SUBUNIT(EC 3.5.1.5) 19 20 RXA02278 GR00656 3420 4268 UREASE OPERON URED PROTEIN21 22 RXA02275 GR00656 1632 2102 UREASE ACCESSORY PROTEIN UREE 23 24RXA02276 GR00656 2105 2782 UREASE ACCESSORY PROTEIN UREF 25 26 RXA02277GR00656 2802 3416 UREASE ACCESSORY PROTEIN UREG 27 28 RXA02603 GR007427742 8737 4-HYDROXYBENZOATE OCTAPRENYLTRANSFERASE (EC 2.5.1.—) 29 30RXA01385 GR00406 5320 3440 PHENOL 2 MONOOXYGENASE (EC 1.14.13.7)Proteolysis 31 32 RXN00675 VV0005 33258 34049 METHIONINE AMINOPEPTIDASE(EC 3.4.11.18) 33 34 F RXA00675 GR00178 2 484 METHIONINE AMINOPEPTIDASE(EC 3.4.11.18) 35 36 RXA01609 GR00449 2740 3612 METHIONINEAMINOPEPTIDASE (EC 3.4.11.18) 37 38 RXA01358 GR00393 5337 6857ATP-DEPENDENT PROTEASE LA (EC 3.4.21.53) 39 40 RXA01458 GR00420 32252176 ATP-DEPENDENT PROTEASE LA (EC 3.4.21.53) 41 42 RXA01654 GR00459 9861981 (AL022121) putative alkaline serine protease [Mycobacteriumtuberculosis] 43 44 RXN01868 VV0127 9980 11905 ZINC METALLOPROTEASE (EC3.4.24.—) 45 46 F RXA01868 GR00534 1640 30 ZINC METALLOPROTEASE (EC3.4.24.—) 47 48 F RXA01869 GR00534 1954 1652 ZINC METALLOPROTEASE (EC3.4.24.—) 49 50 RXN03028 VV0008 41156 43930 ATP-DEPENDENT CLP PROTEASEATP-BINDING SUBUNIT CLPA 51 52 F RXA02470 GR00715 2216 3196ATP-DEPENDENT CLP PROTEASE ATP-BINDING SUBUNIT CLPA 53 54 F RXA02471GR00715 3159 4991 ATP-DEPENDENT CLP PROTEASE ATP-BINDING SUBUNIT CLPA 5556 RXA02630 GR00748 2654 1332 (AL021999) putative serine protease[Mycobacterium tuberculosis] 57 58 RXA02834 GR00823 3 497 ATPases withchaperone activity, ATP-dependent protease subunit 59 60 RXA00112GR00016 3687 2497 PROBABLE PERIPLASMIC SERINE PROTEASE DO-LIKE PRECURSOR61 62 RXA00566 GR00152 742 137 ATP-DEPENDENT CLP PROTEASE PROTEOLYTICSUBUNIT (EC 3.4.21.92) 63 64 RXA00567 GR00152 1388 798 ATP-DEPENDENT CLPPROTEASE PROTEOLYTIC SUBUNIT (EC 3.4.21.92) 65 66 RXN03094 VV0057 179443 CLPB PROTEIN 67 68 F RXA01668 GR00464 2205 3920 CLPB PROTEIN 69 70RXN01120 VV0182 5678 4401 ATP-DEPENDENT CLP PROTEASE ATP-BINDING SUBUNITCLPX 71 72 F RXA01120 GR00310 2349 1072 ATP-DEPENDENT CLP PROTEASEATP-BINDING SUBUNIT CLPX 73 74 RXA00744 GR00202 10722 9781 Periplasmicserine proteases 75 76 RXA00844 GR00228 3620 4453 Hypothetical SecretorySerine Protease (EC 3.4.21.—) 77 78 RXA01151 GR00324 862 5 ATP-dependentZn proteases 79 80 RXA02317 GR00665 9664 9053 PEPTIDASE E (EC 3.4.—.—)81 82 RXA02644 GR00751 767 117 XAA-PRO DIPEPTIDASE (EC 3.4.13.9) 83 84RXN02820 VV0131 4799 6109 GAMMA-GLUTAMYLTRANSPEPTIDASE (EC 2.3.2.2) 8586 F RXA02820 GR00801 1 507 GAMMA-GLUTAMYLTRANSPEPTIDASE (EC 2.3.2.2) 8788 F RXA02000 GR00589 3430 3933 GAMMA-GLUTAMYLTRANSPEPTIDASE (EC2.3.2.2) 89 90 RXN03178 VV0334 921 121 PENICILLIN-BINDING PROTEIN 5*PRECURSOR (D-ALANYL-D-ALANINE CARBOXYPEPTIDASE) (EC 3.4.16.4) 91 92 FRXA02859 GR10005 846 121 PENICILLIN-BINDING PROTEIN 5* PRECURSOR(D-ALANYL-D-ALANINE CARBOXYPEPTIDASE) (EC 3.4.16.4) 93 94 RXA00137GR00022 738 1826 XAA-PRO AMINOPEPTIDASE (EC 3.4.11.9) 95 96 RXN00499VV0086 8158 9438 PROLINE IMINOPEPTIDASE (EC 3.4.11.5) 97 98 F RXA00499GR00125 3 959 PROLINE IMINOPEPTIDASE 99 100 RXN00877 VV0099 2221 3885PEPTIDYL-DIPEPTIDASE DCP (EC 3.4.15.5) 101 102 F RXA00877 GR00242 3 1067PEPTIDYL-DIPEPTIDASE DCP (EC 3.4.15.5) 103 104 RXN01014 VV0209 1332810728 AMINOPEPTIDASE N (EC 3.4.11.2) 105 106 F RXA01014 GR00289 3 1580AMINOPEPTIDASE N (EC 3.4.11.2) 107 108 F RXA01018 GR00290 2289 3152AMINOPEPTIDASE N (EC 3.4.11.2) 109 110 RXA01147 GR00323 1353 94 VACUOLARAMINOPEPTIDASE I PRECURSOR (EC 3.4.11.1) 111 112 RXA01161 GR00329 1253117 XAA-PRO AMINOPEPTIDASE (EC 3.4.11.9) 113 114 RXN01181 VV0065 1 957AMINOPEPTIDASE A/I (EC 3.4.11.1) 115 116 F RXA01181 GR00337 1 957AMINOPEPTIDASE 117 118 RXN01277 VV0009 32155 34158 PROLYL ENDOPEPTIDASE(EC 3.4.21.26) 119 120 F RXA01277 GR00368 1738 50 PROLYL ENDOPEPTIDASE(EC 3.4.21.26) 121 122 RXA01914 GR00548 125 550 AMINOPEPTIDASE 123 124RXA02048 GR00624 207 1580 AMINOPEPTIDASE N (EC 3.4.11.2) 125 126RXN00621 VV0135 5853 5071 PROTEASE II (EC 3.4.21.83) 127 128 F RXA00621GR00163 4075 4857 PTRB periplasmic protease 129 130 RXN00622 VV0135 51503735 PROTEASE II (EC 3.4.21.83) 131 132 F RXA00622 GR00163 4778 6193PTRB periplasmic protease 133 134 RXN00982 VV0149 7596 6091 (L42758)proteinase [Streptomyces lividans] 135 136 F RXA00977 GR00275 1647 2660(L42758) proteinase [Streptomyces lividans] 137 138 F RXA00982 GR002765194 4949 (L42758) proteinase [Streptomyces lividans] 139 140 RXA00152GR00023 7175 5880 HFLC PROTEIN (EC 3.4.—.—) 141 142 RXA02558 GR007314939 3965 HFLC PROTEIN (EC 3.4.—.—) 143 144 RXA00500 GR00125 969 1643O-SIALOGLYCOPROTEIN ENDOPEPTIDASE (EC 3.4.24.57) 145 146 RXA00501GR00125 1643 2149 O-SIALOGLYCOPROTEIN ENDOPEPTIDASE (EC 3.4.24.57) 147148 RXA00502 GR00125 2156 3187 O-SIALOGLYCOPROTEIN ENDOPEPTIDASE (EC3.4.24.57) Enzymes in general 149 150 RXN02589 VV0098 16346 17110Hypothetical Methyltransferase (EC 2.1.1.—) 151 152 F RXA02589 GR0074113804 13040 Predicted S-adenosylmethionine-dependent methyltransferase153 154 RXA00226 GR00032 26836 26012 SAM-dependent methyltransferases155 156 RXN01885 VV0184 2004 2804 Hypothetical Methyltransferase (EC2.1.1.—) 157 158 F RXA01885 GR00539 1589 2389 SAM-dependentmethyltransferases 159 160 RXA02592 GR00741 18477 17707 SAM-dependentmethyltransferases 161 162 RXN01795 VV0093 722 1318 MODIFIKATIONMETHYLASE (EC 2.1.1.73) 163 164 F RXA01795 GR00507 706 1140 MODIFICATIONMETHYLASE (EC 2.1.1.73) 165 166 RXA01214 GR00351 1640 3130 LACCASE 1PRECURSOR (EC 1.10.3.2) 167 168 RXA01250 GR00364 592 5 LACCASE 1PRECURSOR (EC 1.10.3.2) 169 170 RXA02477 GR00715 10581 11201 CARBONICANHYDRASE (EC 4.2.1.1) 171 172 RXN00833 GR00225 374 6 THIOL PEROXIDASE(EC 1.11.1.—) 173 174 F RXA00833 GR00225 374 6 THIOL PEROXIDASE (EC1.11.1.—) 175 176 RXA01224 GR00354 4186 5208 2-NITROPROPANE DIOXYGENASE(EC 1.13.11.32) 177 178 RXA01182 GR00337 1363 971 HypotheticalOxidoreductase 179 180 RXA02531 GR00726 1226 1936 HypotheticalOxidoreductase 181 182 RXN00689 VV0005 22416 20926 BETAINE-ALDEHYDEDEHYDROGENASE PRECURSOR (EC 1.2.1.8) 183 184 F RXA00689 GR00180 1401 775BETAINE-ALDEHYDE DEHYDROGENASE PRECURSOR (EC 1.2.1.8) 185 186 RXN03128VV0120 3 857 MORPHINE 6-DEHYDROGENASE (EC 1.1.1.218) 187 188 F RXA02192GR00643 2 523 MORPHINE 6-DEHYDROGENASE (EC 1.1.1.218) 189 190 RXA02351GR00679 132 1070 NITRILOTRIACETATE MONOOXYGENASE COMPONENT A (EC1.14.13.—) 191 192 RXN00905 VV0238 8075 8875 N-ACYL-L-AMINO ACIDAMIDOHYDROLASE (EC 3.5.1.14) 193 194 F RXA00905 GR00247 2 694N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 195 196 RXA00906GR00247 630 1133 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 197198 RXA00907 GR00247 1143 1265 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC3.5.1.14) 199 200 RXA02101 GR00631 3104 1842 N-ACYL-L-AMINO ACIDAMIDOHYDROLASE (EC 3.5.1.14) 201 202 RXN02565 VV0154 14299 13034N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 203 204 F RXA02565GR00733 1 342 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 205 206 FRXA02567 GR00734 3 740 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14)207 208 RXN03077 VV0043 1729 2913 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC3.5.1.14) 209 210 F RXA02855 GR10002 1693 2877 N-ACYL-L-AMINO ACIDAMIDOHYDROLASE (EC 3.5.1.14), hippurate hydrolase 211 212 RXA00026GR00003 3657 5042 Hypothetical Amidohydrolase (EC 3.5.1.—) 213 214RXA01971 GR00569 963 133 Hypothetical Metal-Dependent Hydrolase 215 216RXA01802 GR00509 3461 4291 Predicted hydrolases (HAD superfamily) 217218 RXN00866 VV0258 3557 4522 Predicted Zn-dependent hydrolases 219 220F RXA00866 GR00236 3555 4499 Predicted Zn-dependent hydrolases 221 222RXA02410 GR00703 792 127 Predicted Zn-dependent hydrolases 223 224RXA00961 GR00267 2 433 SALICYLATE HYDROXYLASE (EC 1.14.13.1) 225 226RXA00111 GR00016 930 1922 SOLUBLE EPOXIDE HYDROLASE (SEH) (EC 3.3.2.3)227 228 RXA01932 GR00555 6479 5583 ACETYL-HYDROLASE (EC 3.1.1.—) 229 230RXA02574 GR00739 833 1840 PUTATIVE SECRETED HYDROLASE 231 232 RXN00983VV0231 1796 321 SIALIDASE PRECURSOR (EC 3.2.1.18) 233 234 F RXA00983GR00278 1200 4 SIALIDASE PRECURSOR (EC 3.2.1.18) 235 236 RXA00984GR00278 1716 1300 SIALIDASE PRECURSOR (EC 3.2.1.18) 237 238 RXN02513VV0193 737 6 SIALIDASE PRECURSOR (EC 3.2.1.18) 239 240 F RXA02513GR00722 93 824 SIALIDASE PRECURSOR (EC 3.2.1.18) 241 242 RXA00903GR00246 637 5 Putative epimerase 243 244 RXA01224 GR00354 4186 52082-NITROPROPANE DIOXYGENASE (EC 1.13.11.32) 245 246 RXA01571 GR00438 13601959 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) 247 248 RXN02478 VV0119 75646350 SIALIDASE PRECURSOR (EC 3.2.1.18) 249 250 RXN00343 VV0125 1118 63-OXOSTEROID 1-DEHYDROGENASE (EC 1.3.99.4) 251 252 RXN01555 VV0135 2982028861 3-OXOSTEROID 1-DEHYDROGENASE (EC 1.3.99.4) 253 254 RXN01166 VV011718142 16838 EXTRACELLULAR LIPASE PRECURSOR (EC 3.1.1.3) 255 256 RXN02001VV0326 630 1787 N-ACYL-L-AMINO ACID AMIDOHYDROLASE (EC 3.5.1.14) 257 258RXN03145 VV0142 7561 7115 4-OXALOCROTONATE TAUTOMERASE (EC 5.3.2.—) 259260 RXN01466 VV0019 7050 6091 ARYLESTERASE (EC 3.1.1.2) 261 262 RXN01145VV0077 7538 6525 KETOL-ACID REDUCTOISOMERASE (EC 1.1.1.86) 263 264RXN03088 VV0052 3431 3817 Hypothetical Methyltransferase (EC 2.1.1.—)265 266 RXN02952 VV0320 1032 1547 PUTATIVE REDUCTASE 267 268 RXN00513VV0092 1573 653 CARBOXYVINYL-CARBOXYPHOSPHONATE PHOSPHORYLMUTASE (EC2.7.8.23) 269 270 RXN01152 VV0136 1740 907 PROTEIN-L-ISOASPARTATEO-METHYLTRANSFERASE (EC 2.1.1.77) 271 272 RXN00787 VV0321 3736 5637D-AMINO ACID DEHYDROGENASE LARGE SUBUNIT (EC 1.4.99.1) N-metabolism 273274 RXN01302 VV0148 2837 2385 NITRATE REDUCTASE ALPHA CHAIN (EC1.7.99.4) 275 276 F RXA01302 GR00376 370 5 NITRATE REDUCTASE ALPHA CHAIN(EC 1.7.99.4) 277 278 RXN01308 VV0148 2406 4 NITRATE REDUCTASE ALPHACHAIN (EC 1.7.99.4) 279 280 F RXA01307 GR00377 686 6 NITRATE REDUCTASEALPHA CHAIN (EC 1.7.99.4) 281 282 F RXA01308 GR00378 1211 6 NITRATEREDUCTASE ALPHA CHAIN (EC 1.7.99.4) 283 284 RXN01309 VV0158 1 801NITRATE REDUCTASE ALPHA CHAIN (EC 1.7.99.4) 285 286 F RXA01309 GR00379719 51 NITRATE REDUCTASE ALPHA CHAIN (EC 1.7.99.4) 287 288 RXA02017GR00610 1731 1048 NITRATE REDUCTASE ALPHA CHAIN (EC 1.7.99.4) 289 290RXA02018 GR00610 2788 1739 NITRATE REDUCTASE BETA CHAIN (EC 1.7.99.4)291 292 RXA02016 GR00610 1036 260 NITRATE REDUCTASE GAMMA CHAIN (EC1.7.99.4) 293 294 RXA00471 GR00119 2997 3686 NITRATE/NITRITE RESPONSEREGULATOR PROTEIN NARL 295 296 RXA00133 GR00021 201 1013 NITRATE/NITRITERESPONSE REGULATOR PROTEIN NARP 297 298 RXA00650 GR00169 4017 3382NITRATE/NITRITE RESPONSE REGULATOR PROTEIN NARP 299 300 RXA01189 GR003392545 1937 NITRATE/NITRITE RESPONSE REGULATOR PROTEIN NARP 301 302RXA01607 GR00449 123 752 NITRATE/NITRITE RESPONSE REGULATOR PROTEIN NARP303 304 RXN00470 VV0086 27401 28669 NITRATE/NITRITE SENSOR PROTEIN NARX(EC 2.7.3.—) 305 306 F RXA00470 GR00119 1752 2951 NITRATE/NITRITE SENSORPROTEIN NARX (EC 2.7.3.—) 307 308 RXA00756 GR00203 2932 1937 NUTILIZATION SUBSTANCE PROTEIN A 309 310 RXA00139 GR00022 2514 3224 NUTILIZATION SUBSTANCE PROTEIN B 311 312 RXA01303 GR00376 1724 390NITRITE EXTRUSION PROTEIN 313 314 RXA01412 GR00412 620 417 NITROGENFIXATION PROTEIN FIXI (PROBABLE E1-E2 TYPE CATION ATPASE) (EC 3.6.1.—)315 316 RXA00773 GR00205 3208 4350 NITROGEN REGULATION PROTEIN NIFR3 317318 RXA02746 GR00764 1 267 NITROGEN REGULATORY PROTEIN P-II 319 320RXA02745 GR00763 15350 14472 NODULATION ATP-BINDING PROTEIN I 321 322RXN00820 VV0054 19455 19817 NODULATION PROTEIN N 323 324 F RXA00820GR00221 1007 1369 NODULATION PROTEIN N 325 326 RXA01059 GR00296 87829390 OXYGEN-INSENSITIVE NAD(P)H NITROREDUCTASE (EC 1.—.—.—) 327 328RXN01386 VV0008 39246 38317 NITRILASE REGULATOR 329 330 RXN00073 VV01542369 687 FERREDOXIN-NITRITE REDUCTASE (EC 1.7.7.1) 331 332 RXN03131VV0127 276 4 RHIZOPINE CATABOLISM PROTEIN MOCC 333 334 RXS00153 VV01674195 4620 NODULATION PROTEIN Urease Phosphate and Phosphonate metabolism335 336 RXN01716 VV0319 3259 2774 EXOPOLYPHOSPHATASE (EC 3.6.1.11) 337338 RXN02972 VV0319 2763 2353 EXOPOLYPHOSPHATASE (EC 3.6.1.11) 339 340RXN00663 VV0142 10120 11493 PHOH PROTEIN HOMOLOG 341 342 RXN00778 VV010318126 19250 PHOSPHATE-BINDING PERIPLASMIC PROTEIN PRECURSOR 343 344RXN00250 VV0189 286 1032 DEDA PROTEIN - ALKALINE PHOSPHATASE LIKEPROTEIN Sulfate metabolism 345 346 RXA00072 GR00012 446 6PHOSPHOADENOSINE PHOSPHOSULFATE REDUCTASE (EC 1.8.99.4) 347 348 RXA00793GR00211 1469 2644 SULFATE STARVATION-INDUCED PROTEIN 6 349 350 RXA01192GR00342 161 733 SULFATE STARVATION-INDUCED PROTEIN 6 351 352 RXA00715GR00188 2120 2914 THIOSULFATE SULFURTRANSFERASE (EC 2.8.1.1) 353 354RXA01664 GR00463 1306 485 THIOSULFATE SULFURTRANSFERASE (EC 2.8.1.1) 355356 RXN02334 VV0141 7939 7217 THIOSULFATE SULFURTRANSFERASE (EC 2.8.1.1)357 358 F RXA02334 GR00672 2 355 THIOSULFATE SULFURTRANSFERASE (EC2.8.1.1) Fe-Metabolism 359 360 RXN01499 VV0008 7034 3213 ENTEROBACTINSYNTHETASE COMPONENT F 361 362 RXN01997 VV0084 33308 33793 FERRITIN MgMetabolism 363 364 RXA01848 GR00524 1532 789 MAGNESIUM-CHELATASE SUBUNITCHLI 365 366 RXN01849 VV0139 16415 17515 MAGNESIUM-CHELATASE SUBUNITCHLI 367 368 F RXA01849 GR00524 2004 1555 MAGNESIUM-CHELATASE SUBUNITCHLI 369 370 F RXA01691 GR00474 570 4 MAGNESIUM-CHELATASE SUBUNIT CHLI371 372 RXN00665 VV0252 135 635 MG2+/CITRATE COMPLEX SECONDARYTRANSPORTER Modification and degradation of aromatic compounds 373 374RXN03026 VV0007 28635 28901 3-DEHYDROQUINATE DEHYDRATASE (EC 4.2.1.10)375 376 RXN02908 VV0025 8507 8247 O-SUCCINYLBENZOIC ACID—COA LIGASE (EC6.2.1.26) 377 378 RXN03000 VV0235 570 4 SALICYLATE HYDROXYLASE (EC1.14.13.1) 379 380 RXN03036 VV0014 671 6 PROTOCATECHUATE 3,4-DIOXYGENASEBETA CHAIN (EC 1.13.11.3) 381 382 RXN02974 VV0229 12631 124374-NITROPHENYLPHOSPHATASE (EC 3.1.3.41) 383 384 RXN00393 VV0025 7241 63481,4-DIHYDROXY-2-NAPHTHOATE OCTAPRENYLTRANSFERASE (EC 2.5.—.—) 385 386RXN00948 VV0107 4266 5384 12-oxophytodienoate reductase (EC 1.3.1.42)387 388 RXN01923 VV0020 3384 41332-HYDROXY-6-OXO-6-PHENYLHEXA-2,4-DIENOATE HYDROLASE (EC 3.7.1.—) 389 390RXN00398 VV0025 14633 13884 2-PYRONE-4,6-DICARBOXYLATE LACTONASE (EC3.1.1.57) 391 392 RXN02813 VV0128 13120 14118 3-CARBOXY-CIS,CIS-MUCONATECYCLOISOMERASE HOMOLOG (EC 5.5.1.2) 393 394 RXN00136 VV0134 13373 144673-DEHYDROQUINATE SYNTHASE (EC 4.6.1.3) 395 396 RXN02508 VV0007 2673328586 3-DEHYDROSHIKIMATE DEHYDRATASE (EC 4.2.1.—) 397 398 RXN02839VV0362 3 449 4-HYDROXYBENZOATE OCTAPRENYLTRANSFERASE (EC 2.5.1.—) 399400 RXN00639 VV0128 7858 8712 CATECHOL 1,2-DIOXYGENASE (EC 1.13.11.1)401 402 RXN02530 VV0057 5469 6125 DIMETHYLANILINE MONOOXYGENASE (N-OXIDEFORMING) 1 (EC 1.14.13.8) 403 404 RXN00434 VV0112 12078 11212 QUINONEOXIDOREDUCTASE (EC 1.6.5.5) 405 406 RXN01619 VV0050 24649 23675 QUINONEOXIDOREDUCTASE (EC 1.6.5.5) 407 408 RXN01842 VV0234 1615 2532 QUINONEOXIDOREDUCTASE (EC 1.6.5.5) 409 410 RXN00641 VV0128 7440 5950 TOLUATE1,2-DIOXYGENASE ALPHA SUBUNIT (EC 1.14.12.—) 411 412 RXN01993 VV0182 161143 VANILLATE DEMETHYLASE (EC 1.14.—.—) 413 414 RXN00658 VV0083 1570516397 PHENOL 2-MONOOXYGENASE (EC 1.14.13.7) 415 416 RXN00178 VV017414670 15554 hydroxyquinol 1,2-dioxygenase (EC 1.13.11.37) 417 418RXN01461 VV0128 12414 13025 PROTOCATECHUATE 3,4-DIOXYGENASE ALPHA CHAIN(EC 1.13.11.3) 419 420 RXN01653 VV0321 12867 11407 DIBENZOTHIOPHENEDESULFURIZATION ENZYME A 421 422 RXN02053 VV0009 39448 40026 DRGAPROTEIN 423 424 RXN00177 VV0174 13589 14656 MALEYLACETATE REDUCTASE (EC1.3.1.32) 425 426 RXC00963 VV0249 1816 2652 PROTEIN involved indegradation of aromatic compounds Modification and degradation ofaliphatic compounds 427 428 RXN00299 VV0176 43379 42402 ALKANALMONOOXYGENASE ALPHA CHAIN (EC 1.14.14.3) 429 430 F RXA00299 GR00048 73766633 ALKANAL MONOOXYGENASE ALPHA CHAIN (EC 1.14.14.3) 431 432 RXA00332GR00057 16086 15385 ALKANAL MONOOXYGENASE ALPHA CHAIN (EC 1.14.14.3) 433434 RXA01838 GR00519 2 820 ALKANAL MONOOXYGENASE ALPHA CHAIN (EC1.14.14.3) 435 436 RXA02643 GR00750 1603 560 ALKANAL MONOOXYGENASE ALPHACHAIN (EC 1.14.14.3) 437 438 RXA01933 GR00555 6590 7192 2-HALOALKANOICACID DEHALOGENASE I (EC 3.8.1.2) 439 440 RXA02351 GR00679 132 1070NITRILOTRIACETATE MONOOXYGENASE COMPONENT A (EC 1.14.13.—)

TABLE 2 GENES IDENTIFIED FROM GENBANK GenBank ™ Accession No. Gene NameGene Function Reference A09073 ppg Phosphoenol pyruvate carboxylaseBachmann, B. et al. “DNA fragment coding for phosphoenolpyruvatcorboxylase, recombinant DNA carrying said fragment, strains carryingthe recombinant DNA and method for producing L-aminino acids using saidstrains,” Patent: EP 0358940-A 3 Mar. 21, 1990 A45579, Threoninedehydratase Moeckel, B. et al. “Production of L-isoleucine by means ofrecombinant A45581, micro-organisms with deregulated threoninedehydratase,” Patent: WO A45583, 9519442-A 5 Jul. 20, 1995 A45585 A45587AB003132 murC; ftsQ; ftsZ Kobayashi, M. et al. “Cloning, sequencing, andcharacterization of the ftsZ gene from coryneform bacteria,” Biochem.Biophys. Res. Commun., 236(2): 383-388 (1997) AB015023 murC; ftsQ Wachi,M. et al. “A murC gene from Coryneform bacteria,” Appl. Microbiol.Biotechnol., 51(2): 223-228 (1999) AB018530 dtsR Kimura, E. et al.“Molecular cloning of a novel gene, dtsR, which rescues the detergentsensitivity of a mutant derived from Brevibacterium lactofermentum,”Biosci. Biotechnol. Biochem., 60(10): 1565-1570 (1996) AB018531 dtsR1;dtsR2 AB020624 murI D-glutamate racemase AB023377 tkt transketolaseAB024708 gltB; gltD Glutamine 2-oxoglutarate aminotransferase large andsmall subunits AB025424 acn aconitase AB027714 rep Replication proteinAB027715 rep; aad Replication protein; aminoglycoside adenyltransferaseAF005242 argC N-acetylglutamate-5-semialdehyde dehydrogenase AF005635glnA Glutamine synthetase AF030405 hisF cyclase AF030520 argGArgininosuccinate synthetase AF031518 argF Ornithinecarbamolytransferase AF036932 aroD 3-dehydroquinate dehydratase AF038548pyc Pyruvate carboxylase AF038651 dciAE; apt; rel Dipeptide-bindingprotein; adenine Wehmeier, L. et al. “The role of the Corynebacteriumglutamicum rel gene in phosphoribosyltransferase; GTP (p)ppGppmetabolism,” Microbiology, 144: 1853-1862 (1998) pyrophosphokinaseAF041436 argR Arginine repressor AF045998 impA Inositol monophosphatephosphatase AF048764 argH Argininosuccinate lyase AF049897 argC; argJ;argB; argD; argF; N-acetylglutamylphosphate reductase; argR; argG; argHornithine acetyltransferase; N- acetylglutamate kinase; acetylornithinetransminase; ornithine carbamoyltransferase; arginine repressor;argininosuccinate synthase; argininosuccinate lyase AF050109 inhAEnoyl-acyl carrier protein reductase AF050166 hisG ATPphosphoribosyltransferase AF051846 hisAPhosphoribosylformimino-5-amino-1- phosphoribosyl-4-imidazolecarboxamideisomerase AF052652 metA Homoserine O-acetyltransferase Park, S. et al.“Isolation and analysis of metA, a methionine biosynthetic gene encodinghomoserine acetyltransferase in Corynebacterium glutamicum,” Mol.Cells., 8(3): 286-294 (1998) AF053071 aroB Dehydroquinate synthetaseAF060558 hisH Glutamine amidotransferase AF086704 hisEPhosphoribosyl-ATP- pyrophosphohydrolase AF114233 aroA5-enolpyruvylshikimate 3-phosphate synthase AF116184 panDL-aspartate-alpha-decarboxylase precursor Dusch, N. et al. “Expressionof the Corynebacterium glutamicum panD gene encodingL-aspartate-alpha-decarboxylase leads to pantothenate overproduction inEscherichia coli,” Appl. Environ. Microbiol., 65(4)1530-1539 (1999)AF124518 aroD; aroE 3-dehydroquinase; shikimate dehydrogenase AF124600aroC; aroK; aroB; Chorismate synthase; shikimate kinase; 3- pepQdehydroquinate synthase; putative cytoplasmic peptidase AF145897 inhAAF145898 inhA AJ001436 ectP Transport of ectoine, glycine betaine,Peter, H. et al. “Corynebacterium glutamicum is equipped with foursecondary proline carriers for compatible solutes: Identification,sequencing, and characterization of the proline/ectoine uptake system,ProP, and the ectoine/proline/glycine betaine carrier, EctP,” J.Bacteriol., 180(22): 6005-6012 (1998) AJ004934 dapDTetrahydrodipicolinate succinylase Wehrmann, A. et al. “Different modesof diaminopimelate synthesis and their (incomplete^(i)) role in cellwall integrity: A study with Corynebacterium glutamicum,” J. Bacteriol.,180(12): 3159-3165 (1998) AJ007732 ppc; secG; amt; ocd;Phosphoenolpyruvate-carboxylase; ?; high soxA affinity ammonium uptakeprotein; putative ornithine-cyclodecarboxylase; sarcosine oxidaseAJ010319 ftsY, glnB, glnD; srp; Involved in cell division; PII protein;Jakoby, M. et al. “Nitrogen regulation in Corynebacterium glutamicum;amtP uridylyltransferase (uridylyl-removing Isolation of genes involvedin biochemical characterization of corresponding enzmye); signalrecognition particle; low proteins,” FEMS Microbiol., 173(2): 303-310(1999) affinity ammonium uptake protein AJ132968 cat Chloramphenicolaceteyl transferase AJ224946 mqo L-malate: quinone oxidoreductaseMolenaar, D. et al. “Biochemical and genetic characterization of themembrane-associated malate dehydrogenase (acceptor) from Corynebacteriumglutamicum,” Eur. J. Biochem., 254(2): 395-403 (1998) AJ238250 ndh NADHdehydrogenase AJ238703 porA Porin Lichtinger, T. et al. “Biochemical andbiophysical characterization of the cell wall porin of Corynebacteriumglutamicum: The channel is formed by a low molecular mass polypeptide,”Biochemistry, 37(43): 15024-15032 (1998) D17429 Transposable elementIS31831 Vertes, A. A. et al. “Isolation and characterization of IS31831,a transposable element from Corynebacterium glutamicum,” Mol.Microbiol., 11(4): 739-746 (1994) D84102 odhA 2-oxoglutaratedehydrogenase Usuda, Y. et al. “Molecular cloning of the Corynebacteriumglutamicum (Brevibacterium lactofermentum AJ12036) odhA gene encoding anovel type of 2-oxoglutarate dehydrogenase,” Microbiology, 142:3347-3354 (1996) E01358 hdh; hk Homoserine dehydrogenase; homoserineKatsumata, R. et al. “Production of L-thereonine and L-isoleucine,”Patent: JP kinase 1987232392-A 1 Oct. 12, 1987 E01359 Upstream of thestart codon of homoserine Katsumata, R. et al. “Production ofL-thereonine and L-isoleucine,” Patent: JP kinase gene 1987232392-A 2Oct. 12, 1987 E01375 Tryptophan operon E01376 trpL; trpE Leader peptide;anthranilate synthase Matsui, K. et al. “Tryptophan operon, peptide andprotein coded thereby, utilization of tryptophan operon gene expressionand production of tryptophan,” Patent: JP 1987244382-A 1 Oct. 24, 1987E01377 Promoter and operator regions of Matsui, K. et al. “Tryptophanoperon, peptide and protein coded thereby, tryptophan operon utilizationof tryptophan operon gene expression and production of tryptophan,”Patent: JP 1987244382-A 1 Oct. 24, 1987 E03937 Biotin-synthaseHatakeyama, K. et al. “DNA fragment containing gene capable of codingbiotin synthetase and its utilization,” Patent: JP 1992278088-A 1 Oct.02, 1992 E04040 Diamino pelargonic acid aminotransferase Kohama, K. etal. “Gene coding diaminopelargonic acid aminotransferase anddesthiobiotin synthetase and its utilization,” Patent: JP 1992330284-A 1Nov. 18, 1992 E04041 Desthiobiotinsynthetase Kohama, K. et al. “Genecoding diaminopelargonic acid aminotransferase and desthiobiotinsynthetase and its utilization,” Patent: JP 1992330284-A 1 Nov. 18, 1992E04307 Flavum aspartase Kurusu, Y. et al. “Gene DNA coding aspartase andutilization thereof,” Patent: JP 1993030977-A 1 Feb. 09, 1993 E04376Isocitric acid lyase Katsumata, R. et al. “Gene manifestationcontrolling DNA,” Patent: JP 1993056782-A 3 Mar. 09, 1993 E04377Isocitric acid lyase N-terminal fragment Katsumata, R. et al. “Genemanifestation controlling DNA,” Patent: JP 1993056782-A 3 Mar. 09, 1993E04484 Prephenate dehydratase Sotouchi, N. et al. “Production ofL-phenylalanine by fermentation,” Patent: JP 1993076352-A 2 Mar. 30,1993 E05108 Aspartokinase Fugono, N. et al. “Gene DNA codingAspartokinase and its use,” Patent: JP 1993184366-A 1 Jul. 27, 1993E05112 Dihydro-dipichorinate synthetase Hatakeyama, K. et al. “Gene DNAcoding dihydrodipicolinic acid synthetase and its use,” Patent: JP1993184371-A 1 Jul. 27, 1993 E05776 Diaminopimelic acid dehydrogenaseKobayashi, M. et al. “Gene DNA coding Diaminopimelic acid dehydrogenaseand its use,” Patent: JP 1993284970-A 1 Nov. 02, 1993 E05779 Threoninesynthase Kohama, K. et al. “Gene DNA coding threonine synthase and itsuse,” Patent: JP 1993284972-A 1 Nov. 02, 1993 E06110 Prephenatedehydratase Kikuchi, T. et al. “Production of L-phenylalanine byfermentation method,” Patent: JP 1993344881-A 1 Dec. 27, 1993 E06111Mutated Prephenate dehydratase Kikuchi, T. et al. “Production ofL-phenylalanine by fermentation method,” Patent: JP 1993344881-A 1 Dec.27, 1993 E06146 Acetohydroxy acid synthetase Inui, M. et al. “Genecapable of coding Acetohydroxy acid synthetase and its use,” Patent: JP1993344893-A 1 Dec. 27, 1993 E06825 Aspartokinase Sugimoto, M. et al.“Mutant aspartokinase gene,” patent: JP 1994062866-A 1 Mar. 08, 1994E06826 Mutated aspartokinase alpha subunit Sugimoto, M. et al. “Mutantaspartokinase gene,” patent: JP 1994062866-A 1 Mar. 08, 1994 E06827Mutated aspartokinase alpha subunit Sugimoto, M. et al. “Mutantaspartokinase gene,” patent: JP 1994062866-A 1 Mar. 08, 1994 E07701 secYHonno, N. et al. “Gene DNA participating in integration of membraneousprotein to membrane,” Patent: JP 1994169780-A 1 Jun. 21, 1994 E08177Aspartokinase Sato, Y. et al. “Genetic DNA capable of codingAspartokinase released from feedback inhibition and its utilization,”Patent: JP 1994261766-A 1 Sep. 20, 1994 E08178, Feedbackinhibition-released Aspartokinase Sato, Y. et al. “Genetic DNA capableof coding Aspartokinase released from E08179, feedback inhibition andits utilization,” Patent: JP 1994261766-A 1 Sep. 20, 1994 E08180,E08181, E08182 E08232 Acetohydroxy-acid isomeroreductase Inui, M. et al.“Gene DNA coding acetohydroxy acid isomeroreductase,” Patent: JP1994277067-A 1 Oct. 04, 1994 E08234 secE Asai, Y. et al. “Gene DNAcoding for translocation machinery of protein,” Patent: JP 1994277073-A1 Oct. 04, 1994 E08643 FT aminotransferase and desthiobiotin Hatakeyama,K. et al. “DNA fragment having promoter function in synthetase promoterregion coryneform bacterium,” Patent: JP 1995031476-A 1 Feb. 03, 1995E08646 Biotin synthetase Hatakeyama, K. et al. “DNA fragment havingpromoter function in coryneform bacterium,” Patent: JP 1995031476-A 1Feb. 03, 1995 E08649 Aspartase Kohama, K. et al “DNA fragment havingpromoter function in coryneform bacterium,” Patent: JP 1995031478-A 1Feb. 03, 1995 E08900 Dihydrodipicolinate reductase Madori, M. et al.“DNA fragment containing gene coding Dihydrodipicolinate acid reductaseand utilization thereof,” Patent: JP 1995075578-A 1 Mar. 20, 1995 E08901Diaminopimelic acid decarboxylase Madori, M. et al. “DNA fragmentcontaining gene coding Diaminopimelic acid decarboxylase and utilizationthereof,” Patent: JP 1995075579-A 1 Mar. 20, 1995 E12594 Serinehydroxymethyltransferase Hatakeyama, K. et al. “Production ofL-trypophan,” Patent: JP 1997028391-A 1 Feb. 4, 1997 E12760, transposaseMoriya, M. et al. “Amplification of gene using artificial transposon,”Patent: E12759, JP 1997070291-A Mar. 18, 1997 E12758 E12764 Arginyl-tRNAsynthetase; diaminopimelic Moriya, M. et al. “Amplification of geneusing artificial transposon,” Patent: acid decarboxylase JP 1997070291-AMar. 18, 1997 E12767 Dihydrodipicolinic acid synthetase Moriya, M. etal. “Amplification of gene using artificial transposon,” Patent: JP1997070291-A Mar. 18, 1997 E12770 aspartokinase Moriya, M. et al.“Amplification of gene using artificial transposon,” Patent: JP1997070291-A Mar. 18, 1997 E12773 Dihydrodipicolinic acid reductaseMoriya, M. et al. “Amplification of gene using artificial transposon,”Patent: JP 1997070291-A Mar. 18, 1997 E13655 Glucose-6-phosphatedehydrogenase Hatakeyama, K. et al. “Glucose-6-phosphate dehydrogenaseand DNA capable of coding the same,” Patent: JP 1997224661-A 1 Sep. 02,1997 L01508 IlvA Threonine dehydratase Moeckel, B. et al. “Functionaland structural analysis of the threonine dehydratase of Corynebacteriumglutamicum,” J. Bacteriol., 174: 8065-8072 (1992) L07603 EC 4.2.1.153-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. “The cloning andnucleotide sequence of Corynebacterium phosphate synthase glutamicum3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene,” FEMSMicrobiol. Lett., 107: 223-230 (1993) L09232 IlvB; ilvN; ilvCAcetohydroxy acid synthase large subunit; Keilhauer, C. et al.“Isoleucine synthesis in Corynebacterium glutamicum: Acetohydroxy acidsynthase small subunit; molecular analysis of the ilvB-ilvN-ilvCoperon,” J. Bacteriol., 175(17): 5595-5603 Acetohydroxy acidisomeroreductase (1993) L18874 PtsM Phosphoenolpyruvate sugar Fouet, Aet al. “Bacillus subtilis sucrose-specific enzyme II of thephosphotransferase phosphotransferase system: expression in Escherichiacoli and homology to enzymes II from enteric bacteria,” PNAS USA,84(24): 8773-8777 (1987); Lee, J. K. et al. “Nucleotide sequence of thegene encoding the Corynebacterium glutamicum mannose enzyme II andanalyses of the deduced protein sequence,” FEMS Microbiol. Lett.,119(1-2): 137-145 (1994) L27123 aceB Malate synthase Lee, H-S. et al.“Molecular characterization of aceB, a gene encoding malate synthase inCorynebacterium glutamicum,” J. Microbiol. Biotechnol., 4(4): 256-263(1994) L27126 Pyruvate kinase Jetten, M. S. et al. “Structural andfunctional analysis of pyruvate kinase from Corynebacterium glutamicum,”Appl. Environ. Microbiol., 60(7): 2501-2507 (1994) L28760 aceAIsocitrate lyase L35906 dtxr Diphtheria toxin repressor Oguiza, J. A. etal. “Molecular cloning, DNA sequence analysis, and characterization ofthe Corynebacterium diphtheriae dtxR from Brevibacteriumlactofermentum,” J. Bacteriol., 177(2): 465-467 (1995) M13774 Prephenatedehydratase Follettie, M. T. et al. “Molecular cloning and nucleotidesequence of the Corynebacterium glutamicum pheA gene,” J. Bacteriol.,167: 695-702 (1986) M16175 5S rRNA Park, Y-H. et al. “Phylogeneticanalysis of the coryneform bacteria by 56 rRNA sequences,” J.Bacteriol., 169: 1801-1806 (1987) M16663 trpE Anthranilate synthase, 5′end Sano, K. et al. “Structure and function of the trp operon controlregions of Brevibacterium lactofermentum, a glutamic-acid-producingbacterium,” Gene, 52: 191-200 (1987) M16664 trpA Tryptophan synthase,3′end Sano, K. et al. “Structure and function of the trp operon controlregions of Brevibacterium lactofermentum, a glutamic-acid-producingbacterium,” Gene, 52: 191-200 (1987) M25819 Phosphoenolpyruvatecarboxylase O'Regan, M. et al. “Cloning and nucleotide sequence of thePhosphoenolpyruvate carboxylase-coding gene of Corynebacteriumglutamicum ATCC13032,” Gene, 77(2): 237-251 (1989) M85106 23S rRNA geneinsertion sequence Roller, C. et al. “Gram-positive bacteria with a highDNA G + C content are characterized by a common insertion within their23S rRNA genes,” J. Gen. Microbiol., 138: 1167-1175 (1992) M85107, 23SrRNA gene insertion sequence Roller, C. et al. “Gram-positive bacteriawith a high DNA G + C content are M85108 characterized by a commoninsertion within their 23S rRNA genes,” J. Gen. Microbiol., 138:1167-1175 (1992) M89931 aecD; brnQ; yhbw Beta C-S lyase; branched-chainamino acid Rossol, I. et al. “The Corynebacterium glutamicum aecD geneencodes a C-S uptake carrier; hypothetical protein yhbw lyase withalpha, beta-elimination activity that degrades aminoethylcysteine,” J.Bacteriol., 174(9): 2968-2977 (1992); Tauch, A. et al. “Isoleucineuptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQgene product,” Arch. Microbiol., 169(4): 303-312 (1998) S59299 trpLeader gene (promoter) Herry, D. M. et al. “Cloning of the trp genecluster from a tryptophan-hyperproducing strain of Corynebacteriumglutamicum: identification of a mutation in the trp leader sequence,”Appl. Environ. Microbiol., 59(3): 791-799 (1993) U11545 trpDAnthranilate phosphoribosyltransferase O'Gara, J. P. and Dunican, L. K.(1994) Complete nucleotide sequence of the Corynebacterium glutamicumATCC 21850 tpD gene.” Thesis, Microbiology Department, UniversityCollege Galway, Ireland. U13922 cglIM; cglIR; clgIIR Putative type II5-cytosoine Schafer, A. et al. “Cloning and characterization of a DNAregion encoding a methyltransferase; putative type II stress-sensitiverestriction system from Corynebacterium glutamicum ATCC restrictionendonuclease; putative type I or 13032 and analysis of its role inintergeneric conjugation with Escherichia type III restrictionendonuclease coli,” J. Bacteriol., 176(23): 7309-7319 (1994); Schafer,A. et al. “The Corynebacterium glutamicum cglIM gene encoding a5-cytosine in an McrBC- deficient Escherichia coli strain,” Gene,203(2): 95-101 (1997) U14965 recA U31224 ppx Ankri, S. et al. “Mutationsin the Corynebacterium glutamicumproline biosynthetic pathway: A naturalbypass of the proA step,” J. Bacteriol., 178(15): 4412-4419 (1996)U31225 proC L-proline: NADP+ 5-oxidoreductase Ankri, S. et al.“Mutations in the Corynebacterium glutamicumproline biosyntheticpathway: A natural bypass of the proA step,” J. Bacteriol., 178(15):4412-4419 (1996) U31230 obg; proB; unkdh ?; gamma glutamyl kinase;similar to D- Ankri, S. et al. “Mutations in the Corynebacteriumglutamicumproline isomer specific 2-hydroxyacid biosynthetic pathway: Anatural bypass of the proA step,” J. Bacteriol., dehydrogenases 178(15):4412-4419 (1996) U31281 bioB Biotin synthase Serebriiskii, I. G., “Twonew members of the bio B superfamily: Cloning, sequencing and expressionof bio B genes of Methylobacillus flagellatum and Corynebacteriumglutamicum,” Gene, 175: 15-22 (1996) U35023 thtR; accBC Thiosulfatesulfurtransferase; acyl CoA Jager, W. et al. “A Corynebacteriumglutamicum gene encoding a two-domain carboxylase protein similar tobiotin carboxylases and biotin-carboxyl-carrier proteins,” Arch.Microbiol., 166(2); 76-82 (1996) U43535 cmr Multidrug resistance proteinJager, W. et al. “A Corynebacterium glutamicum gene conferring multidrugresistance in the heterologous host Escherichia coli,” J. Bacteriol.,179(7): 2449-2451 (1997) U43536 clpB Heat shock ATP-binding proteinU53587 aphA-3 3′5″-aminoglycoside phosphotransferase U89648Corynebacterium glutamicum unidentified sequence involved in histidinebiosynthesis, partial sequence X04960 trpA; trpB; trpC; trpD; Tryptophanoperon Matsui, K. et al. “Complete nucleotide and deduced amino acidsequences of trpE; trpG; trpL the Brevibacterium lactofermentumtryptophan operon,” Nucleic Acids Res., 14(24): 10113-10114 (1986)X07563 lys A DAP decarboxylase (meso-diaminopimelate Yeh, P. et al.“Nucleic sequence of the lysA gene of Corynebacterium decarboxylase, EC4.1.1.20) glutamicum and possible mechanisms for modulation of itsexpression,” Mol. Gen. Genet., 212(1): 112-119 (1988) X14234 EC 4.1.1.31Phosphoenolpyruvate carboxylase Eikmanns, B. J. et al. “ThePhosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum:Molecular cloning, nucleotide sequence, and expression,” Mol. Gen.Genet., 218(2): 330-339 (1989); Lepiniec, L. et al. “SorghumPhosphoenolpyruvate carboxylase gene family: structure, function andmolecular evolution,” Plant. Mol. Biol., 21 (3): 487-502 (1993) X17313fda Fructose-bisphosphate aldolase Von der Osten, C. H. et al.“Molecular cloning, nucleotide sequence and fine- structural analysis ofthe Corynebacterium glutamicum fda gene: structural comparison of C.glutamicum fructose-1,6-biphosphate aldolase to class I and class IIaldolases,” Mol. Microbiol., X53993 dapA L-2,3-dihydrodipicolinatesynthetase (EC Bonnassie, S. et al. “Nucleic sequence of the dapA genefrom 4.2.1.52) Corynebacterium glutamicum,” Nucleic Acids Res., 18(21):6421 (1990) X54223 AttB-related site Cianciotto, N. et al. “DNA sequencehomology between att B-related sites of Corynebacterium diphtheriae,Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP siteof lambdacorynephage,” FEMS. Microbiol, Lett., 66: 299-302 (1990) X54740argS; lysA Arginyl-tRNA synthetase; Diaminopimelate Marcel, T. et al.“Nucleotide sequence and organization of the upstream regiondecarboxylase of the Corynebacterium glutamicum lysA gene,” Mol.Microbiol., 4(11): 1819-1830 (1990) X55994 trpL; trpE Putative leaderpeptide; anthranilate Heery, D. M. et al. “Nucleotide sequence of theCorynebacterium glutamicum synthase component 1 trpE gene,” NucleicAcids Res., 18(23): 7138 (1990) X56037 thrC Threonine synthase Han, K.S. et al. “The molecular structure of the Corynebacterium glutamicumthreonine synthase gene,” Mol. Microbiol., 4(10): 1693-1702 (1990)X56075 attB-related site Attachment site Cianciotto, N. et al. “DNAsequence homology between att B-related sites of Corynebacteriumdiphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, andthe attP site of lambdacorynephage,” FEMS. Microbiol, Lett., 66: 299-302(1990) X57226 lysC-alpha; lysC-beta; Aspartokinase-alpha subunit;Kalinowski, J. et al. “Genetic and biochemical analysis of theAspartokinase asd Aspartokinase-beta subunit; aspartate beta fromCorynebacterium glutamicum,” Mol. Microbiol., 5(5): 1197-1204 (1991);semialdehyde dehydrogenase Kalinowski, J. et al. “Aspartokinase geneslysC alpha and lysC beta overlap and are adjacent to the aspertatebeta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum,”Mol. Gen. Genet., 224(3): 317-324 (1990) X59403 gap; pgk; tpiGlyceraldehyde-3-phosphate; Eikmanns, B. J. “Identification, sequenceanalysis, and expression of a phosphoglycerate kinase; triosephosphateCorynebacterium glutamicum gene cluster encoding the three glycolyticisomerase enzymes glyceraldehyde-3-phosphate dehydrogenase,3-phosphoglycerate kinase, and triosephosphate isomeras,” J. Bacteriol.,174(19): 6076-6086 (1992) X59404 gdh Glutamate dehydrogenase Bormann, E.R. et al. “Molecular analysis of the Corynebacterium glutamicum gdh geneencoding glutamate dehydrogenase,” Mol. Microbiol., 6(3): 317-326 (1992)X60312 lysI L-lysine permease Seep-Feldhaus, A. H. et al. “Molecularanalysis of the Corynebacterium glutamicum lysI gene involved in lysineuptake,” Mol. Microbiol., 5(12): 2995-3005 (1991) X66078 cop1 Ps1protein Joliff, G. et al. “Cloning and nucleotide sequence of the csp1gene encoding PS1, one of the two major secreted proteins ofCorynebacterium glutamicum: The deduced N-terminal region of PS1 issimilar to the Mycobacterium antigen 85 complex,” Mol. Microbiol.,6(16): 2349-2362 (1992) X66112 glt Citrate synthase Eikmanns, B. J. etal. “Cloning sequence, expression and transcriptional analysis of theCorynebacterium glutamicum gltA gene encoding citrate synthase,”Microbiol., 140: 1817-1828 (1994) X67737 dapB Dihydrodipicolinatereductase X69103 csp2 Surface layer protein PS2 Peyret, J. L. et al.“Characterization of the cspB gene encoding PS2, an orderedsurface-layer protein in Corynebacterium glutamicum,” Mol. Microbiol.,9(1): 97-109 (1993) X69104 IS3 related insertion element Bonamy, C. etal. “Identification of IS1206, a Corynebacterium glutamicum IS3-relatedinsertion sequence and phylogenetic analysis,” Mol. Microbiol., 14(3):571-581 (1994) X70959 leuA Isopropylmalate synthase Patek, M. et al.“Leucine synthesis in Corynebacterium glutamicum: enzyme activities,structure of leuA, and effect of leuA inactivation on lysine synthesis,”Appl. Environ. Microbiol., 60(1): 133-140 (1994) X71489 icd Isocitratedehydrogenase (NADP+) Eikmanns, B. J. et al. “Cloning sequence analysis,expression, and inactivation of the Corynebacterium glutamicum icd geneencoding isocitrate dehydrogenase and biochemical characterization ofthe enzyme,” J. Bacteriol., 177(3): 774-782 (1995) X72855 GDHA Glutamatedehydrogenase (NADP+) X75083, mtrA 5-methyltryptophan resistance Heery,D. M. et al. “A sequence from a tryptophan-hyperproducing strain ofX70584 Corynebacterium glutamicum encoding resistance to5-methyltryptophan,” Biochem. Biophys. Res. Commun., 201(3): 1255-1262(1994) X75085 recA Fitzpatrick, R. et al. “Construction andcharacterization of recA mutant strains of Corynebacterium glutamicumand Brevibacterium lactofermentum,” Appl. Microbiol. Biotechnol., 42(4):575-580 (1994) X75504 aceA; thiX Partial Isocitrate lyase; ? Reinscheid,D. J. et al. “Characterization of the isocitrate lyase gene fromCorynebacterium glutamicum and biochemical analysis of the enzyme,” J.Bacteriol., 176(12): 3474-3483 (1994) X76875 ATPase beta-subunit Ludwig,W. et al. “Phylogenetic relationships of bacteria based on comparativesequence analysis of elongation factor Tu and ATP-synthase beta-subunitgenes,” Antonie Van Leeuwenhoek, 64: 285-305 (1993) X77034 tufElongation factor Tu Ludwig, W. et al. “Phylogenetic relationships ofbacteria based on comparative sequence analysis of elongation factor Tuand ATP-synthase beta-subunit genes,” Antonie Van Leeuwenhoek, 64:285-305 (1993) X77384 recA Billman-Jacobe, H. “Nucleotide sequence of arecA gene from Corynebacterium glutamicum,” DNA Seq., 4(6): 403-404(1994) X78491 aceB Malate synthase Reinscheid, D. J. et al. “Malatesynthase from Corynebacterium glutamicum pta-ack operon encodingphosphotransacetylase: sequence analysis,” Microbiology, 140: 3099-3108(1994) X80629 16S rDNA 16S ribosomal RNA Rainey, F. A. et al.“Phylogenetic analysis of the genera Rhodococcus and Norcardia andevidence for the evolutionary origin of the genus Norcardia from withinthe radiation of Rhodococcus species,” Microbiol., 141: 523-528 (1995)X81191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, W. et al.“Structure of the gluABCD cluster encoding the gluD glutamate uptakesystem of Corynebacterium glutamicum,” J. Bacteriol., 177(5): 1152-1158(1995) X81379 dapE Succinyldiaminopimelate desuccinylase Wehrmann, A. etal. “Analysis of different DNA fragments of Corynebacterium glutamicumcomplementing dapE of Escherichia coli,” Microbiology, 40: 3349-56(1994) X82061 16S rDNA 16S ribosomal RNA Ruimy, R. et al. “Phylogeny ofthe genus Corynebacterium deduced from analyses of small-subunitribosomal DNA sequences,” Int. J. Syst. Bacteriol., 45(4): 740-746(1995) X82928 asd; lysC Aspartate-semialdehyde dehydrogenase; ?Serebrijski, I. et al. “Multicopy suppression by asd gene and osmoticstress- dependent complementation by heterologous proA in proA mutants,”J. Bacteriol., 177(24): 7255-7260 (1995) X82929 proA Gamma-glutamylphosphate reductase Serebrijski, I. et al. “Multicopy suppression by asdgene and osmotic stress- dependent complementation by heterologous proAin proA mutants,” J. Bacteriol., 177(24): 7255-7260 (1995) X84257 16SrDNA 16S ribosomal RNA Pascual, C. et al. “Phylogenetic analysis of thegenus Corynebacterium based on 16S rRNA gene sequences,” Int. J. Syst.Bacteriol., 45(4): 724-728 (1995) X85965 aroP; dapE Aromatic amino acidpermease; ? Wehrmann, A. et al. “Functional analysis of sequencesadjacent to dapE of Corynebacterium glutamicumproline reveals thepresence of aroP, which encodes the aromatic amino acid transporter,” J.Bacteriol., 177(20): 5991-5993 (1995) X86157 argB; argC; argD;Acetylglutamate kinase; N-acetyl-gamma- Sakanyan, V. et al. “Genes andenzymes of the acetyl cycle of arginine argF; argJ glutamyl-phosphatereductase; biosynthesis in Corynebacterium glutamicum: enzyme evolutionin the early acetylornithine aminotransferase; ornithine steps of thearginine pathway,” Microbiology, 142: 99-108 (1996)carbamoyltransferase; glutamate N- acetyltransferase X89084 pta; ackAPhosphate acetyltransferase; acetate kinase Reinscheid, D. J. et al.“Cloning, sequence analysis, expression and inactivation of theCorynebacterium glutamicum pta-ack operon encoding phosphotransacetylaseand acetate kinase,” Microbiology, 145: 503-513 (1999) X89850 attBAttachment site Le Marrec, C. et al. “Genetic characterization ofsite-specific integration functions of phi AAU2 infecting “Arthrobacteraureus C70,” J. Bacteriol., 178(7): 1996-2004 (1996) X90356 Promoterfragment F1 Patek, M. et al. “Promoters from Corynebacterium glutamicum:cloning, molecular analysis and search for a consensus motif,”Microbiology, 142: 1297-1309 (1996) X90357 Promoter fragment F2 Patek,M. et al. “Promoters from Corynebacterium glutamicum: cloning, molecularanalysis and search for a consensus motif,” Microbiology, 142: 1297-1309(1996) X90358 Promoter fragment F10 Patek, M. et al. “Promoters fromCorynebacterium glutamicum: cloning, molecular analysis and search for aconsensus motif,” Microbiology, 142: 1297-1309 (1996) X90359 Promoterfragment F13 Patek, M. et al. “Promoters from Corynebacteriumglutamicum: cloning, molecular analysis and search for a consensusmotif,” Microbiology, 142: 1297-1309 (1996) X90360 Promoter fragment F22Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,molecular analysis and search for a consensus motif,” Microbiology, 142:1297-1309 (1996) X90361 Promoter fragment F34 Patek, M. et al.“Promoters from Corynebacterium glutamicum: cloning, molecular analysisand search for a consensus motif,” Microbiology, 142: 1297-1309 (1996)X90362 Promoter fragment F37 Patek, M. et al. “Promoters fromCorynebacterium glutamicum: cloning, molecular analysis and search for aconsensus motif,” Microbiology, 142: 1297-1309 (1996) X90363 Promoterfragment F45 Patek, M. et al. “Promoters from Corynebacteriumglutamicum: cloning, molecular analysis and search for a consensusmotif,” Microbiology, 142: 1297-1309 (1996) X90364 Promoter fragment F64Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,molecular analysis and search for a consensus motif,” Microbiology, 142:1297-1309 (1996) X90365 Promoter fragment F75 Patek, M. et al.“Promoters from Corynebacterium glutamicum: cloning, molecular analysisand search for a consensus motif,” Microbiology, 142: 1297-1309 (1996)X90366 Promoter fragment PF101 Patek, M. et al. “Promoters fromCorynebacterium glutamicum: cloning, molecular analysis and search for aconsensus motif,” Microbiology, 142: 1297-1309 (1996) X90367 Promoterfragment PF104 Patek, M. et al. “Promoters from Corynebacteriumglutamicum: cloning, molecular analysis and search for a consensusmotif,” Microbiology, 142: 1297-1309 (1996) X90368 Promoter fragmentPF109 Patek, M. et al. “Promoters from Corynebacterium glutamicum:cloning, molecular analysis and search for a consensus motif,”Microbiology, 142: 1297-1309 (1996) X93513 amt Ammonium transport systemSiewe, R. M. et al. “Functional and genetic characterization of the(methyl) ammonium uptake carrier of Corynebacterium glutamicum,” J.Biol. Chem., 271(10): 5398-5403 (1996) X93514 betP Glycine betainetransport system Peter, H. et al. “Isolation, characterization, andexpression of the Corynebacterium glutamicum betP gene, encoding thetransport system for the compatible solute glycine betaine,” J.Bacteriol., 178(17): 5229-5234 (1996) X95649 orf4 Patek, M. et al.“Identification and transcriptional analysis of the dapB-ORF2- dapA-ORF4operon of Corynebacterium glutamicum, encoding two enzymes involved inL-lysine synthesis,” Biotechnol. Lett., 19: 1113-1117 (1997) X96471lysE; lysG Lysine exporter protein; Lysine export Vrljic, M. et al. “Anew type of transporter with a new type of cellular regulator proteinfunction: L-lysine export from Corynebacterium glutamicum,” Mol.Microbiol., 22(5): 815-826 (1996) X96580 panB; panC; xylB3-methyl-2-oxobutanoate Sahm, H. et al. “D-pantothenate synthesis inCorynebacterium glutamicum and hydroxymethyltransferase; pantoate-beta-use of panBC and genes encoding L-valine synthesis for D-pantothenatealanine ligase; xylulokinase overproduction,” Appl. Environ. Microbiol.,65(5): 1973-1979 (1999) X96962 Insertion sequence IS1207 and transposaseX99289 Elongation factor P Ramos, A. et al. “Cloning, sequencing andexpression of the gene encoding elongation factor P in the amino-acidproducer Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC13869),” Gene, 198: 217-222 (1997) Y00140 thrB Homoserine kinase Mateos,L. M. et al. “Nucleotide sequence of the homoserine kinase (thrB) geneof the Brevibacterium lactofermentum,” Nucleic Acids Res., 15(9): 3922(1987) Y00151 ddh Meso-diaminopimelate D-dehydrogenase Ishino, S. et al.“Nucleotide sequence of the meso-diaminopimelate D- (EC 1.4.1.16)dehydrogenase gene from Corynebacterium glutamicum,” Nucleic Acids Res.,15(9): 3917 (1987) Y00476 thrA Homoserine dehydrogenase Mateos, L. M. etal. “Nucleotide sequence of the homoserine dehydrogenase (thrA) gene ofthe Brevibacterium lactofermentum,” Nucleic Acids Res., 15(24): 10598(1987) Y00546 hom; thrB Homoserine dehydrogenase; homoserine Peoples, O.P. et al. “Nucleotide sequence and fine structural analysis of thekinase Corynebacterium glutamicum hom-thrB operon,” Mol. Microbiol.,2(1): 63-72 (1988) Y08964 murC; ftsQ/divD; ftsZUPD-N-acetylmuramate-alanine ligase; Honrubia, M. P. et al.“Identification, characterization, and chromosomal division initiationprotein or cell division organization of the ftsZ gene fromBrevibacterium lactofermentum,” Mol. Gen. protein; cell division proteinGenet., 259(1): 97-104 (1998) Y09163 putP High affinity prolinetransport system Peter, H. et al. “Isolation of the putP gene ofCorynebacterium glutamicumproline and characterization of a low-affinityuptake system for compatible solutes,” Arch. Microbiol., 168(2): 143-151(1997) Y09548 pyc Pyruvate carboxylase Peters-Wendisch, P. G. et al.“Pyruvate carboxylase from Corynebacterium glutamicum: characterization,expression and inactivation of the pyc gene,” Microbiology, 144: 915-927(1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek, M. et al.“Analysis of the leuB gene from Corynebacterium glutamicum,” Appl.Microbiol. Biotechnol., 50(1): 42-47 (1998) Y12472 Attachment sitebacteriophage Phi-16 Moreau, S. et al. “Site-specific integration ofcorynephage Phi-16: The construction of an integration vector,”Microbiol., 145: 539-548 (1999) Y12537 proP Proline/ectoine uptakesystem protein Peter, H. et al. “Corynebacterium glutamicum is equippedwith four secondary carriers for compatible solutes: Identification,sequencing, and characterization of the proline/ectoine uptake system,ProP, and the ectoine/proline/glycine betaine carrier, EctP,” J.Bacteriol., 180(22): 6005-6012 (1998) Y13221 glnA Glutamine synthetase IJakoby, M. et al. “Isolation of Corynebacterium glutamicum glnA geneencoding glutamine synthetase I,” FEMS Microbiol. Lett., 154(1): 81-88(1997) Y16642 lpd Dihydrolipoamide dehydrogenase Y18059 Attachment siteCorynephage 304L Moreau, S. et al. “Analysis of the integrationfunctions of &phi; 304L: An integrase module among corynephages,”Virology, 255(1): 150-159 (1999) Z21501 argS; lysA Arginyl-tRNAsynthetase; diaminopimelate Oguiza, J. A. et al. “A gene encodingarginyl-tRNA synthetase is located in the decarboxylase (partial)upstream region of the lysA gene in Brevibacterium lactofermentum:Regulation of argS-lysA cluster expression by arginine,” J. Bacteriol.,175(22): 7356-7362 (1993) Z21502 dapA; dapB Dihydrodipicolinatesynthase; Pisabarro, A. et al. “A cluster of three genes (dapA, orf2,and dapB) of dihydrodipicolinate reductase Brevibacterium lactofermentumencodes dihydrodipicolinate reductase, and a third polypeptide ofunknown function,” J. Bacteriol., 175(9): 2743-2749 (1993) Z29563 thrCThreonine synthase Malumbres, M. et al. “Analysis and expression of thethrC gene of the encoded threonine synthase,” Appl. Environ. Microbiol.,60(7)2209-2219 (1994) Z46753 16S rDNA Gene for 16S ribosomal RNA Z49822sigA SigA sigma factor Oguiza, J. A. et al “Multiple sigma factor genesin Brevibacterium lactofermentum: Characterization of sigA and sigB,” J.Bacteriol., 178(2): 550-553 (1996) Z49823 galE; dtxR Catalytic activityUDP-galactose 4- Oguiza, J. A. et al “The galE gene encoding theUDP-galactose 4-epimerase of epimerase; diphtheria toxin regulatoryBrevibacterium lactofermentum is coupled transcriptionally to the dmdRprotein gene,” Gene, 177: 103-107 (1996) Z49824 orfl; sigB ?; SigB sigmafactor Oguiza, J. A. et al “Multiple sigma factor genes inBrevibacterium lactofermentum: Characterization of sigA and sigB,” J.Bacteriol., 178(2): 550-553 (1996) Z66534 Transposase Correia, A. et al.“Cloning and characterization of an IS-like element present in thegenome of Brevibacterium lactofermentum ATCC 13869,” Gene, 170(1): 91-94(1996)¹A sequence for this gene was published in the indicated reference.However, the sequence obtained by the inventors of the presentapplication is significantly longer than the published version. It isbelieved that the published version relied on an incorrect start codon,and thus represents only a fragment of the actual coding region.

TABLE 3 Corynebacterium and Brevibacterium Strains Which May be Used inthe Practice of the Invention Genus species ATCC FERM NRRL CECT NCIMBCBS NCTC DSMZ Brevibacterium ammoniagenes 21054 Brevibacteriumammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacteriumammoniagenes 19352 Brevibacterium ammoniagenes 19353 Brevibacteriumammoniagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacteriumammoniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacteriumammoniagenes 21077 Brevibacterium ammoniagenes 21553 Brevibacteriumammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacteriumbutanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacteriumflavum 21474 Brevibacterium flavum 21129 Brevibacterium flavum 21518Brevibacterium flavum B11474 Brevibacterium flavum B11472 Brevibacteriumflavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427Brevibacterium flavum 21475 Brevibacterium flavum 21517 Brevibacteriumflavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum B11477Brevibacterium flavum B11478 Brevibacterium flavum 21127 Brevibacteriumflavum B11474 Brevibacterium healii 15527 Brevibacterium keto glutamicum21004 Brevibacterium keto glutamicum 21089 Brevibacterium ketosoreductum21914 Brevibacterium lactofermentum 70 Brevibacterium lactofermentum 74Brevibacterium lactofermentum 77 Brevibacterium lactofermentum 21798Brevibacterium lactofermentum 21799 Brevibacterium lactofermentum 21800Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B11470Brevibacterium lactofermentum B11471 Brevibacterium lactofermentum 21086Brevibacterium lactofermentum 21420 Brevibacterium lactofermentum 21086Brevibacterium lactofermentum 31269 Brevibacterium linens 9174Brevibacterium linens 19391 Brevibacterium linens 8377 Brevibacteriumparaffinolyticum 11160 Brevibacterium spec. 717.73 Brevibacterium spec.717.73 Brevibacterium spec. 14604 Brevibacterium spec. 21860Brevibacterium spec. 21864 Brevibacterium spec. 21865 Brevibacteriumspec. 21866 Brevibacterium spec. 19240 Corynebacterium acetoacidophilum21476 Corynebacterium acetoacidophilum 13870 Corynebacterium acetoglutamicum B11473 Corynebacterium aceto glutamicum B11475Corynebacterium aceto glutamicum 15806 Corynebacterium aceto glutamicum21491 Corynebacterium aceto glutamicum 31270 Corynebacterium acetophilumB3671 Corynebacterium ammoniagenes 6872 2399 Corynebacteriumammoniagenes 15511 Corynebacterium fujiokense 21496 Corynebacteriumglutamicum 14067 Corynebacterium glutamicum 39137 Corynebacteriumglutamicum 21254 Corynebacterium glutamicum 21255 Corynebacteriumglutamicum 31830 Corynebacterium glutamicum 13032 Corynebacteriumglutamicum 14305 Corynebacterium glutamicum 15455 Corynebacteriumglutamicum 13058 Corynebacterium glutamicum 13059 Corynebacteriumglutamicum 13060 Corynebacterium glutamicum 21492 Corynebacteriumglutamicum 21513 Corynebacterium glutamicum 21526 Corynebacteriumglutamicum 21543 Corynebacterium glutamicum 13287 Corynebacteriumglutamicum 21851 Corynebacterium glutamicum 21253 Corynebacteriumglutamicum 21514 Corynebacterium glutamicum 21516 Corynebacteriumglutamicum 21299 Corynebacterium glutamicum 21300 Corynebacteriumglutamicum 39684 Corynebacterium glutamicum 21488 Corynebacteriumglutamicum 21649 Corynebacterium glutamicum 21650 Corynebacteriumglutamicum 19223 Corynebacterium glutamicum 13869 Corynebacteriumglutamicum 21157 Corynebacterium glutamicum 21158 Corynebacteriumglutamicum 21159 Corynebacterium glutamicum 21355 Corynebacteriumglutamicum 31808 Corynebacterium glutamicum 21674 Corynebacteriumglutamicum 21562 Corynebacterium glutamicum 21563 Corynebacteriumglutamicum 21564 Corynebacterium glutamicum 21565 Corynebacteriumglutamicum 21566 Corynebacterium glutamicum 21567 Corynebacteriumglutamicum 21568 Corynebacterium glutamicum 21569 Corynebacteriumglutamicum 21570 Corynebacterium glutamicum 21571 Corynebacteriumglutamicum 21572 Corynebacterium glutamicum 21573 Corynebacteriumglutamicum 21579 Corynebacterium glutamicum 19049 Corynebacteriumglutamicum 19050 Corynebacterium glutamicum 19051 Corynebacteriumglutamicum 19052 Corynebacterium glutamicum 19053 Corynebacteriumglutamicum 19054 Corynebacterium glutamicum 19055 Corynebacteriumglutamicum 19056 Corynebacterium glutamicum 19057 Corynebacteriumglutamicum 19058 Corynebacterium glutamicum 19059 Corynebacteriumglutamicum 19060 Corynebacterium glutamicum 19185 Corynebacteriumglutamicum 13286 Corynebacterium glutamicum 21515 Corynebacteriumglutamicum 21527 Corynebacterium glutamicum 21544 Corynebacteriumglutamicum 21492 Corynebacterium glutamicum B8183 Corynebacteriumglutamicum B8182 Corynebacterium glutamicum B12416 Corynebacteriumglutamicum B12417 Corynebacterium glutamicum B12418 Corynebacteriumglutamicum B11476 Corynebacterium glutamicum 21608 Corynebacteriumlilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacteriumspec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 31088Corynebacterium spec. 31089 Corynebacterium spec. 31090 Corynebacteriumspec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 1595420145 Corynebacterium spec. 21857 Corynebacterium spec. 21862Corynebacterium spec. 21863ATCC: American Type Culture Collection, Rockville, MD, USAFERM: Fermentation Research Institute, Chiba, JapanNRRL: ARS Culture Collection, Northern Regional Research Laboratory,Peoria, IL, USACECT: Coleccion Espanola de Cultivos Tipo, Valencia, SpainNCIMB: National Collection of Industrial and Marine Bacteria Ltd.,Aberdeen, UKCBS: Centraalbureau voor Schimmelcultures, Baarn, NLNCTC: National Collection of Type Cultures, London, UKDSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen,Braunschweig, GermanyFor reference see Sugawara, H. et al. (1993) World directory ofcollections of cultures of microorganisms: Bacteria, fungi and yeasts(4^(th) edn), World federation for culture collections world data centeron microorganisms, Saimata, Japen.

TABLE 4 ALIGNMENT RESULTS length % homology Date of ID # (NT) GenbankHit Length Accession Name of Genbank Hit Source of Genbank Hit (GAP)Deposit rxa00026 1509 GB_RO: MMHC310M6 158405 AF109906 Mus musculus MHCclass III region RD gene, partial cds; Bf, C2, G9A, Mus musculus 38,00310-DEC-1998 NG22, G9, HSP70, HSP70, HSC70t, and smRNP genes, completecds; G7A gene, partial cds; and unknown genes. GB_HTG2: AC007029 119007AC007029 Homo sapiens clone DJ0855F16, *** SEQUENCING IN PROGRESS Homosapiens 37,943 7-Apr-99 ***, 1 unordered pieces. GB_HTG2: AC007029119007 AC007029 Homo sapiens clone DJ0855F16, *** SEQUENCING IN PROGRESSHomo sapiens 37,943 7-Apr-99 ***, 1 unordered pieces. rxa00072 rxa001111116 GB_BA1: SAUSIGA 2748 M94370 Stigmatella aurantiaca sigma factor(sigA) gene, complete cds. Stigmatella aurantiaca 40,435 16-Aug-94GB_BA1: SC5B8 28500 AL022374 Streptomyces coelicolor cosmid 5B8.Streptomyces coelicolor 40,090 22-Apr-98 GB_BA2: AE001767 9086 AE001767Thermotoga maritima section 79 of 136 of the complete genome. Thermotogamaritima 35,091 2-Jun-99 rxa00112 1314 GB_EST35: AU075536 418 AU075536AU075536 Rice shoot Oryza sativa cDNA clone S0028_2Z, mRNA Oryza sativa39,423 7-Jul-99 sequence. GB_GSS9: AQ157585 647 AQ157585 nbxb0009B16rCUGI Rice BAC Library Oryza sativa genomic clone Oryza sativa 40,86712-Sep-98 nbxb0009B16r, genomic survey sequence. GB_GSS14: AQ510314 542AQ510314 nbxb0095O05f CUGI Rice BAC Library Oryza sativa genomic cloneOryza sativa 39,372 04-MAY-1999 nbxb0095O05f, genomic survey sequence.rxa00133 936 GB_BA1: SC2G5 38404 AL035478 Streptomyces coelicolor cosmid2G5. Streptomyces coelicolor 41,170 11-Jun-99 GB_EST7: W64291 515 W64291md98h12.r1 Soares mouse embryo NbME13.5 14.5 Mus musculus Mus musculus35,306 10-Jun-96 cDNA clone IMAGE: 386087 5′ similar to gb: L26528 Musmusculus Rab11b mRNA, complete cds (MOUSE);, mRNA sequence. GB_PR3:AC005624 39594 AC005624 Homo sapiens chromosome 19, cosmid R30017,complete sequence. Homo sapiens 39,054 6-Sep-98 rxa00137 1212 GB_BA2:AF124600 4115 AF124600 Corynebacterium glutamicum chorismate synthase(aroC), shikimate Corynebacterium 99,867 04-MAY-1999 kinase (aroK), and3-dehydroquinate synthase (aroB) genes, complete glutamicum cds; andputative cytoplasmic peptidase (pepQ) gene, partial cds. GB_BA1: MTCY15933818 Z83863 Mycobacterium tuberculosis H37Rv complete genome; segmentMycobacterium 40,959 17-Jun-98 111/162. tuberculosis GB_BA1: MT3DEHQ3437 X59509 M. tuberculosis, genes for 3-dehydroquinate synthase and 3-Mycobacterium 52,583 30-Jun-93 dehydroquinase. tuberculosis rxa00139 834GB_BA1: BLELONP 738 X99289 B. lactofermentum gene encoding elongationfactor P. Corynebacterium 100,000 1-Nov-97 glutamicum GB_PL1: SPAC24C938666 Z98601 S. pombe chromosome I cosmid c24C9. Schizosaccharomyces35,230 24-Feb-99 pombe GB_HTG1: CEY102A5_1 110000 Z99711 Caenorhabditiselegans chromosome V clone Y102A5, *** Caenorhabditis elegans 37,775Z99711 SEQUENCING IN PROGRESS ***, in unordered pieces. rxa00152 1419GB_BA1: MTCY277 38300 Z79701 Mycobacterium tuberculosis H37Rv completegenome; segment Mycobacterium 58,500 17-Jun-98 65/162. tuberculosisGB_BA1: MSGY456 37316 AD000001 Mycobacterium tuberculosis sequence fromclone y456. Mycobacterium 38,913 03-DEC-1996 tuberculosis GB_BA2:AF002133 15437 AF002133 Mycobacterium avium strain GIR10 transcriptionalregulator (mav81) Mycobacterium avium 64,009 26-MAR-1998 gene, partialcds, aconitase (acn), invasin 1 (inv1), invasin 2 (inv2),transcriptional regulator (moxR), ketoacyl-reductase (fabG), enoyl-reductase (inhA) and ferrochelatase (mav272) genes, complete cds.rxa00226 948 GB_PR3: AC005756 43299 AC005756 Homo sapiens chromosome 19,fosmid 39347, complete sequence. Homo sapiens 36,209 02-OCT-1998GB_GSS5: AQ818463 413 AQ818463 HS_5250_A2_B08_SP6E RPCI-11 Human MaleBAC Library Homo Homo sapiens 37,288 26-Aug-99 sapiens genomic clonePlate = 826 Col = 16 Row = C, genomic survey sequence. GB_GSS5: AQ782337832 AQ782337 HS_3184_B1_H12_T7C CIT Approved Human Genomic Sperm Homosapiens 35,917 2-Aug-99 Library D Homo sapiens genomic clone Plate =3184 Col = 23 Row = P, genomic survey sequence. rxa00249 980 GB_BA2:AF035608 3614 AF035608 Pseudomonas aeruginosa ATP sulfurylase smallsubunit (cysD) and Pseudomonas aeruginosa 50,205 1-Jun-98 ATPsulfurylase GTP-binding subunit/APS kinase (cysN) genes, complete cds.GB_BA1: AB017641 17101 AB017641 Micromonospora griseorubida gene forpolyketide synthase, complete Micromonospora 40,266 2-Apr-99 cds.griseorubida GB_BA2: AF002133 15437 AF002133 Mycobacterium avium strainGIR10 transcriptional regulator (mav81) Mycobacterium avium 38,42926-MAR-1998 gene, partial cds, aconitase (acn), invasin 1 (inv1),invasin 2 (inv2), transcriptional regulator (moxR), ketoacyl-reductase(fabG), enoyl- reductase (inhA) and ferrochelatase (mav272) genes,complete cds. rxa00299 1101 GB_BA2: CORCSLYS 2821 M89931 Corynebacteriumglutamicum beta C-S lyase (aecD) and branched- Corynebacterium 100,0004-Jun-98 chain amino acid uptake carrier (brnQ) genes, complete cds, andglutamicum hypothetical protein Yhbw (yhbw) gene, partial cds. GB_BA1:CGECTP 2719 AJ001436 Corynebacterium glutamicum ectP gene.Corynebacterium 41,143 20-Nov-98 glutamicum GB_BA2: AF181035 5922AF181035 Rhodobacter sphaeroides glycogen utilization operon, completeRhodobacter sphaeroides 36,701 7-Sep-99 sequence. rxa00332 825 GB_BA1:CGTHRC 3120 X56037 Corynebacterium glutamicum thrC gene for threoninesynthase (EC Corynebacterium 37,730 17-Jun-97 4.2.99.2). glutamicumGB_PAT: I09078 3146 I09078 Sequence 4 from Patent WO 8809819. Unknown.38,700 02-DEC-1994 GB_PR3: HSJ333B15 73666 AL109954 Human DNA sequencefrom clone 333B15 on chromosome 20, Homo sapiens 37,203 23-Nov-99complete sequence. rxa00470 1392 GB_PL2: DCPCNAM 865 X62977 D. carotamRNA for proliferating cell nuclear antigen (PCNA). Daucus carota 37,91430-Sep-99 GB_PL2: AC006267 101644 AC006267 Arabidopsis thaliana BACF9M13 from chromosome IV near 21.5 cM, Arabidopsis thaliana 36,15827-Apr-99 complete sequence. GB_BA1: TT10SARNA 721 Y15063 Thermusthermophilus 10Sa RNA gene. Thermus thermophilus 39,494 18-Aug-98rxa00471 813 GB_BA1: SERERYAA 11219 M63676 S. erythraea first ORF oferyA gene, complete cds. Saccharopolyspora 38,781 26-Apr-93 erythraeaGB_PAT: AR049367 11219 AR049367 Sequence 1 from patent U.S. Pat. No.5824513. Unknown. 38,781 29-Sep-99 GB_BA1: SERERYAA 11219 M63676 S.erythraea first ORF of eryA gene, complete cds. Saccharopolyspora 38,20526-Apr-93 erythraea rxa00499 1404 GB_PR4: AC007206 42732 AC007206 Homosapiens chromosome 19, cosmid R27370, complete sequence. Homo sapiens34,982 4-Apr-99 GB_EST26: AI344735 462 AI344735 qp05a10.x1 NCI_CGAP_Kid5Homo sapiens cDNA clone Homo sapiens 42,675 2-Feb-99 IMAGE: 1917114 3′similar to gb: M15800 T-LYMPHOCYTE MATURATION-ASSOCIATED PROTEIN(HUMAN);, mRNA sequence. GB_PR4: AC006479 161837 AC006479 Homo sapiensclone DJ1051J04, complete sequence. Homo sapiens 38,462 11-Nov-99rxa00500 798 GB_PR4: AC006111 190825 AC006111 Homo sapiens chromosome 16clone RPCI-11_461A8, complete Homo sapiens 40,736 3-Jul-99 sequence.GB_HTG2: AF128834 196589 AF128834 Homo sapiens chromosome 8 clone BAC57G24 map 8p12, *** Homo sapiens 34,062 28-Feb-99 SEQUENCING IN PROGRESS***, in unordered pieces. GB_HTG2: AF128834 196589 AF128834 Homo sapienschromosome 8 clone BAC 57G24 map 8p12, *** Homo sapiens 34,062 28-Feb-99SEQUENCING IN PROGRESS ***, in unordered pieces. rxa00501 630 GB_BA1:D86429 5925 D86429 Saccharopolyspora rectivirgula gene forbeta-galactosidase, complete Saccharopolyspora 53,871 09-DEC-1998 cds.rectivirgula GB_HTG1: HS1099D15 1301 AL035456 Homo sapiens chromosome 20clone RP5-1099D15, *** Homo sapiens 33,546 23-Nov-99 SEQUENCING INPROGRESS ***, in unordered pieces. GB_HTG1: HS1099D15 1301 AL035456 Homosapiens chromosome 20 clone RP5-1099D15, *** Homo sapiens 33,54623-Nov-99 SEQUENCING IN PROGRESS ***, in unordered pieces. rxa00502 1155GB_BA2: U00015 42325 U00015 Mycobacterium leprae cosmid B1620.Mycobacterium leprae 34,783 01-MAR-1994 GB_BA1: U00020 36947 U00020Mycobacterium leprae cosmid B229. Mycobacterium leprae 34,90001-MAR-1994 GB_HTG1: HS179I15 210672 Z84464 Homo sapiens chromosome 13clone 179I15, *** SEQUENCING IN Homo sapiens 32,898 22-Jan-97 PROGRESS***, in unordered pieces. rxa00566 729 GB_BA1: MTV008 63033 AL021246Mycobacterium tuberculosis H37Rv complete genome; segment Mycobacterium37,011 17-Jun-98 108/162. tuberculosis GB_BA2: AF071885 2188 AF071885Streptomyces coelicolor ATP-dependent Clp protease proteolyticStreptomyces coelicolor 62,963 29-Jun-99 subunit 1 (clpP1) andATP-dependent Clp protease proteolytic subunit 2 (clpP2) genes, completecds; and ATP-dependent Clp protease ATP-binding subunit Clpx (clpX)gene, partial cds. GB_BA2: AF013216 15742 AF013216 Myxococcus xanthusDog (dog), isocitrate lyase (icl), Mls (mls), Ufo Myxococcus xanthus54,683 28-Jan-98 (ufo), fumarate hydratase (fhy), and proteosome majorsubunit (clpP) genes, complete cds; and acyl-CoA oxidase (aco) gene,partial cds. rxa00567 714 GB_BA1: MTV008 63033 AL021246 Mycobacteriumtuberculosis H37Rv complete genome; segment Mycobacterium 42,09017-Jun-98 108/162. tuberculosis GB_BA1: CGBPHI16 962 Y12472 C.glutamicum DNA, attachment site bacteriophage Phi-16. Corynebacterium40,000 05-MAR-1999 glutamicum GB_BA1: ECOCLPPA 1236 J05534 Escherichiacoli ATP-dependent clp protease proteolytic component Escherichia coli52,119 26-Apr-93 (clpP) gene, complete cds. rxa00621 906 GB_EST1: D36491360 D36491 CELK033GYF Yuji Kohara unpublished cDNA Caenorhabditiselegans Caenorhabditis elegans 40,390 8-Aug-94 cDNA clone yk33g11 5′,mRNA sequence. GB_IN2: CELC16A3 34968 U41534 Caenorhabditis eleganscosmid C16A3. Caenorhabditis elegans 35,477 18-MAY-1999 GB_HTG3:AC009311 160198 AC009311 Homo sapiens clone NH0311L03, *** SEQUENCING INPROGRESS Homo sapiens 38,636 13-Aug-99 ***, 3 unordered pieces. rxa006221539 GB_BA1: AB004795 3039 AB004795 Pseudomonas sp. gene for dipeptidylaminopeptidase, complete cds. Pseudomonas sp. 54,721 5-Feb-99 GB_BA1:MBOPII 2392 D38405 Moraxella lacunata gene for protease II, completecds. Moraxella lacunata 50,167 8-Feb-99 GB_IN2: AF078916 2960 AF078916Trypanosoma brucei brucei oligopeptidase B (opb) gene, completeTrypanosoma brucei 48,076 08-OCT-1999 cds. brucei rxa00650 759 GB_BA2:AF161327 2021 AF161327 Corynebacterium diphtheriae histidine kinase ChrS(chrS) and Corynebacterium 51,319 9-Sep-99 response regulator ChrA(chrA) genes, complete cds. diphtheriae GB_PL2: ATAC006533 99188AC006533 Arabidopsis thaliana chromosome II BAC F20M17 genomic sequence,Arabidopsis thaliana 38,051 26-MAY-1999 complete sequence. GB_PL2:ATAC006533 99188 AC006533 Arabidopsis thaliana chromosome II BAC F20M17genomic sequence, Arabidopsis thaliana 35,403 26-MAY-1999 completesequence. rxa00675 915 GB_BA1: SC3C8 33095 AL023861 Streptomycescoelicolor cosmid 3C8. Streptomyces coelicolor 36,836 15-Jan-99 GB_PR3:AC005736 215441 AC005736 Homo sapiens chromosome 16, BAC clone 462G18(LANL), complete Homo sapiens 42,027 01-OCT-1998 sequence. GB_IN2:AC005719 188357 AC005719 Drosophila melanogaster, chromosome 2L, region38A5-38B4, BAC Drosophila melanogaster 35,531 27-OCT-1999 cloneBACR48M05, complete sequence. rxa00689 1614 GB_PAT: E07294 2975 E07294genomic DNA encoding dehydrogenase of Bacillus Bacillus 45,677 29-Sep-97stearothermophilus. stearothermophilus GB_BA1: BACALDHT 1975 D13846 B.stearothermophilus aldhT gene for aldehyde dehydrogenase, Bacillus45,677 20-Feb-99 complete cds. stearothermophilus GB_BA2: PPU96338 5276U96338 Pseudomonas putida NCIMB 9866 plasmid pRA4000 p-cresolPseudomonas putida 44,317 13-MAY-1999 degradative pathway genes,p-hydroxybenzaldehyde dehydrogenase (pchA), p-cresol methylhydroxylase,cytochrome subunit precursor (pchC), unknown (pchX) and p-cresolmethylhydroxylase, flavoprotein subunit (pchF) genes, complete cds.rxa00715 918 GB_EST30: AI647104 218 AI647104 vn15c01.y1 Stratagene mouseheart (#937316) Mus musculus cDNA Mus musculus 58,511 29-Apr-99 cloneIMAGE: 1021248 5′, mRNA sequence. GB_EST17: AA636159 447 AA636159vn15c01.r1 Stratagene mouse heart (#937316) Mus musculus cDNA Musmusculus 41,195 22-OCT-1997 clone IMAGE: 1021248 5′, mRNA sequence.GB_EST10: AA184468 583 AA184468 mt52h05.r1 Stratagene mouse embryoniccarcinoma (#937317) Mus Mus musculus 40,426 12-Feb-97 musculus cDNAclone IMAGE: 633561 5′ similar to gb: D10918 Mouse mRNA for ubiquitinlike protein, partial sequence (MOUSE);, mRNA sequence. rxa00744 1065GB_HTG3: AC009855 167592 AC009855 Homo sapiens clone 1_C_5, ***SEQUENCING IN PROGRESS ***, Homo sapiens 36,673 3-Sep-99 13 unorderedpieces. GB_HTG3: AC009855 167592 AC009855 Homo sapiens clone 1_C_5, ***SEQUENCING IN PROGRESS ***, Homo sapiens 36,673 3-Sep-99 13 unorderedpieces. GB_PR4: AC005082 169739 AC005082 Homo sapiens clone RG271G13,complete sequence. Homo sapiens 39,557 8-Sep-99 rxa00756 1119 GB_BA1:MLCB596 38426 AL035472 Mycobacterium leprae cosmid B596. Mycobacteriumleprae 54,562 27-Aug-99 GB_GSS12: AQ368028 652 AQ368028 toxb0001N11rCUGITomato BAC Library Lycopersicon esculentum Lycopersicon esculentum42,657 5-Feb-99 genomic clone toxb0001N11r, genomic survey sequence.GB_HTG3: AC008067 151242 AC008067 Homo sapiens clone NH0303104, ***SEQUENCING IN PROGRESS Homo sapiens 37,239 8-Sep-99 ***, 2 unorderedpieces. rxa00773 1266 GB_BA1: MLU15182 40123 U15182 Mycobacterium lepraecosmid B2266. Mycobacterium leprae 36,616 09-MAR-1995 GB_BA1: MSGL611CS37769 L78822 Mycobacterium leprae cosmid L611 DNA sequence.Mycobacterium leprae 35,714 15-Jun-96 GB_GSS14: AQ578181 728 AQ578181nbxb0083P08r CUGI Rice BAC Library Oryza sativa genomic clone Oryzasativa 39,246 2-Jun-99 nbxb0083P08r, genomic survey sequence. rxa007931299 GB_GSS5: AQ769737 519 AQ769737 HS_3160_A2_G04_T7C CIT ApprovedHuman Genomic Sperm Homo sapiens 37,765 28-Jul-99 Library D Homo sapiensgenomic clone Plate = 3160 Col = 8 Row = M, genomic survey sequence.GB_BA1: RTU08434 2400 U08434 Rhizobium trifolii orotatephosphoribosyltransferase (pyrE) and Rhizobium trifolii 40,700 16-Apr-97fructokinase (frk) genes, complete cds. GB_EST31: F33810 243 F33810HSPD27491 HM3 Homo sapiens cDNA clone s3000041E12, mRNA Homo sapiens41,564 13-MAY-1999 sequence. rxa00820 486 GB_PR4: AC005868 96180AC005868 Homo sapiens 12q24.2 PAC RPCI5-944M2 (Roswell Park Cancer Homosapiens 32,298 27-Feb-99 Institute Human PAC Library) complete sequence.GB_EST8: AA000903 396 AA000903 mg38b04.r1 Soares mouse embryo NbME13.514.5 Mus musculus Mus musculus 42,045 18-Jul-96 cDNA clone IMAGE: 4260315′, mRNA sequence. GB_EST25: AI317789 696 AI317789 uj20g09.y1 Suganomouse embryo mewa Mus musculus cDNA clone Mus musculus 38,55717-DEC-1998 IMAGE: 1920544 5′ similar to WP: C13C4.5 CE08130 SUGARTRANSPORTER;, mRNA sequence. rxa00833 618 GB_PH: BPH6589 41489 AJ006589Bacteriophage phi-C31 complete genome. Bacteriophage phi-C31 41,80629-Apr-99 GB_HTG2: AC006887 215801 AC006887 Caenorhabditis elegans cloneY59H11, *** SEQUENCING IN Caenorhabditis elegans 35,798 24-Feb-99PROGRESS ***, 3 unordered pieces. GB_HTG2: AC006887 215801 AC006887Caenorhabditis elegans clone Y59H11, *** SEQUENCING IN Caenorhabditiselegans 35,798 24-Feb-99 PROGRESS ***, 3 unordered pieces. rxa00844 957GB_GSS15: AQ605195 459 AQ605195 HS_2136_B1_C12_T7C CIT Approved HumanGenomic Sperm Homo sapiens 38,074 10-Jun-99 Library D Homo sapiensgenomic clone Plate = 2136 Col = 23 Row = F, genomic survey sequence.GB_HTG1: CNS00M8S 214599 AL079302 Homo sapiens chromosome 14 cloneR-1089B7, *** SEQUENCING Homo sapiens 38,120 15-OCT-1999 IN PROGRESS***, in ordered pieces. GB_HTG1: CNS00M8S 214599 AL079302 Homo sapienschromosome 14 clone R-1089B7, *** SEQUENCING Homo sapiens 38,12015-OCT-1999 IN PROGRESS ***, in ordered pieces. rxa00866 1066 GB_BA1:CGORF4GEN 2398 X95649 C. glutamicum ORF4 gene. Corynebacterium 99,27310-MAR-1998 glutamicum GB_BA1: BLDAPAB 3572 Z21502 B. lactofermentumdapA and dapB genes for dihydrodipicolinate Corynebacterium 99,30116-Aug-93 synthase and dihydrodipicolinate reductase. glutamicum GB_PAT:E14517 1411 E14517 DNA encoding Brevibacterium dihydrodipicolinic acidreductase. Corynebacterium 99,659 28-Jul-99 glutamicum rxa00877 1788GB_PAT: I92050 567 I92050 Sequence 17 from patent U.S. Pat. No. 5726299.Unknown. 62,787 01-DEC-1998 GB_PAT: I78760 567 I78760 Sequence 16 frompatent U.S. Pat. No. 5693781. Unknown. 62,787 3-Apr-98 GB_BA2: AE00042610240 AE000426 Escherichia coli K-12 MG1655 section 316 of 400 of thecomplete Escherichia coli 36,456 12-Nov-98 genome. rxa00903 733 GB_BA2:AE001598 11136 AE001598 Chlamydia pneumoniae section 14 of 103 of thecomplete genome. Chlamydophila 32,782 08-MAR-1999 pneumoniae GB_PL2:AF079370 2897 AF079370 Kluyveromyces lactis invertase (INV1) gene,complete cds. Kluyveromyces lactis 35,849 4-Aug-99 GB_BA2: AE00159811136 AE001598 Chlamydia pneumoniae section 14 of 103 of the completegenome. Chlamydophila 40,138 08-MAR-1999 pneumoniae rxa00905 924 GB_PR2:HSQ15C24 73192 AJ239325 Homo sapiens chromosome 21 from cosmids LLNLc1161C16 and Homo sapiens 35,076 28-Sep-99 LLNLc116 15C24 map 21q22.3 regionD21S171-LA161, complete sequence. GB_GSS4: AQ691923 446 AQ691923HS_5400_B2_G04_SP6E RPCI-11 Human Male BAC Library Homo Homo sapiens33,500 6-Jul-99 sapiens genomic clone Plate = 976 Col = 8 Row = N,genomic survey sequence. GB_EST37: AI967802 479 AI967802Ljirnpest12-930-d6 Ljirnp Lambda HybriZap two-hybrid library Lotus Lotusjaponicus 41,127 24-Aug-99 japonicus cDNA clone LP930-12-d6 5′ similarto 60S ribosomal protein L7A, mRNA sequence. rxa00906 627 GB_PAT: I78750588 I78750 Sequence 6 from patent U.S. Pat. No. 5693781. Unknown. 97,0713-Apr-98 GB_PAT: I92039 588 I92039 Sequence 6 from patent U.S. Pat. No.5726299. Unknown. 97,071 01-DEC-1998 GB_PR3: HS929C8 139190 AL020994Human DNA sequence from clone 929C8 on chromosome 22q12.1-12.3 Homosapiens 39,016 23-Nov-99 Contains CA repeat, GSS, STS, completesequence. rxa00907 246 GB_PAT: I78750 588 I78750 Sequence 6 from patentU.S. Pat. No. 5693781. Unknown. 97,561 3-Apr-98 GB_PAT: I92039 588I92039 Sequence 6 from patent U.S. Pat. No. 5726299. Unknown. 97,56101-DEC-1998 GB_PAT: I78750 588 I78750 Sequence 6 from patent U.S. Pat.No. 5693781. Unknown. 37,222 3-Apr-98 rxa00961 455 GB_BA1: AB032799 9077AB032799 Chromobacterium violaceum violacein biosynthetic gene cluster(vio Chromobacterium 39,868 02-OCT-1999 A, vio B, vio C, vio D),complete cds. violaceum GB_BA2: AF172851 10094 AF172851 Chromobacteriumviolaceum violacein biosynthetic gene cluster, Chromobacterium 42,76030-Aug-99 complete sequence. violaceum GB_BA1: AB032799 9077 AB032799Chromobacterium violaceum violacein biosynthetic gene cluster (vioChromobacterium 39,551 02-OCT-1999 A, vio B, vio C, vio D), completecds. violaceum rxa00982 1629 GB_BA1: BLARGS 2501 Z21501 B.lactofermentum argS and lysA genes for arginyl-tRNA synthetaseCorynebacterium 39,003 28-DEC-1993 and diaminopimelate decarboxylase(partial). glutamicum GB_BA1: CGXLYSA 2344 X54740 Corynebacteriumglutamicum argS-lysA operon gene for the upstream Corynebacterium 41,43530-Jun-93 region of the arginyl-tRNA synthetase and diaminopimelateglutamicum decarboxylase (EC 4.1.1.20). GB_PAT: E14508 3579 E14508 DNAencoding Brevibacterium diaminopimelic acid decarboxylase andCorynebacterium 40,566 28-Jul-99 arginyl-tRNA synthase. glutamicumrxa00983 1599 GB_HTG2: AC008152 24000 AC008152 Leishmania majorchromosome 35 clone L7936 strain Friedlin, *** Leishmania major 38,65828-Jul-99 SEQUENCING IN PROGRESS ***, 4 unordered pieces. GB_HTG2:AC008152 24000 AC008152 Leishmania major chromosome 35 clone L7936strain Friedlin, *** Leishmania major 38,658 28-Jul-99 SEQUENCING INPROGRESS ***, 4 unordered pieces. GB_HTG3: AC008648 87249 AC008648 Homosapiens chromosome 5 clone CIT978SKB_186E14, *** Homo sapiens 36,1023-Aug-99 SEQUENCING IN PROGRESS ***, 22 unordered pieces. rxa00984 440GB_BA1: MVINED 3098 D01045 Micromonospora viridifaciens DNA for nedRprotein and Micromonospora 59,226 2-Feb-99 neuraminidase, complete cds.viridifaciens GB_PAT: E02375 1881 E02375 Neuraminidase gene.Micromonospora 59,226 29-Sep-97 viridifaciens GB_PR4: HUAC004513 101311AC004513 Homo sapiens Chromosome 16 BAC clone CIT987SK-A-926E7, Homosapiens 41,204 23-Nov-99 complete sequence. rxa01014 2724 GB_BA1: MTV00863033 AL021246 Mycobacterium tuberculosis H37Rv complete genome; segmentMycobacterium 56,167 17-Jun-98 108/162. tuberculosis GB_BA1: STMAMPEPN2849 L23172 Streptomyces lividans aminopeptidase N gene, complete cds.Streptomyces lividans 57,067 18-MAY-1994 GB_BA1: SC7H2 42655 AL109732Streptomyces coelicolor cosmid 7H2. Streptomyces coelicolor 37,5512-Aug-99 A3(2) rxa01059 732 GB_HTG3: AC008154 172241 AC008154 Homosapiens chromosome 7, *** SEQUENCING IN PROGRESS ***, Homo sapiens39,499 8-Sep-99 26 unordered pieces. GB_HTG3: AC008154 172241 AC008154Homo sapiens chromosome 7, *** SEQUENCING IN PROGRESS ***, Homo sapiens39,499 8-Sep-99 26 unordered pieces. GB_EST32: AI756574 299 AI756574ea02f10.y1 Eimeria M5-6 Merozoite stage Eimeria tenella cDNA 5′, Eimeriatenella 37,793 23-Jun-99 mRNA sequence. rxa01073 954 GB_BA1: BACOUTB1004 M15811 Bacillus subtilis outB gene encoding a sporulation protein,complete Bacillus subtilis 53,723 26-Apr-93 cds. GB_PR4: AC007938 167237AC007938 Homo sapiens clone UWGC: djs201 from 7q31, complete sequence.Homo sapiens 34,322 1-Jul-99 GB_PL2: ATAC006282 92577 AC006282Arabidopsis thaliana chromosome II BAC F13K3 genomic sequence,Arabidopsis thaliana 36,181 13-MAR-1999 complete sequence. rxa01120 1401GB_BA1: MTV008 63033 AL021246 Mycobacterium tuberculosis H37Rv completegenome; segment Mycobacterium 36,715 17-Jun-98 108/162. tuberculosisGB_BA1: CAJ10321 6710 AJ010321 Caulobacter crescentus partial tig geneand clpP, cicA, clpX, lon Caulobacter crescentus 63,311 01-OCT-1998genes. GB_BA2: AF150957 4440 AF150957 Azospirillum brasilense triggerfactor (tig), heat-shock protein ClpP Azospirillum brasilense 60,6137-Jun-99 (clpP), and heat-shock protein ClpX (clpX) genes, complete cds;and Lon protease (lon) gene, partial cds. rxa01147 1383 GB_PR3: HS408N2397916 Z98048 Human DNA sequence from PAC 408N23 on chromosome 22q13.Homo sapiens 34,567 23-Nov-99 Contains HIP, HSC70-INTERACTING PROTEIN(PROGESTERONE RECEPTOR-ASSOCIATED P48 PROTEIN), ESTs and STS. GB_BA2:AE001227 26849 AE001227 Treponema pallidum section 43 of 87 of thecomplete genome. Treponema pallidum 37,564 16-Jul-98 GB_PR3: HS408N2397916 Z98048 Human DNA sequence from PAC 408N23 on chromosome 22q13.Homo sapiens 34,911 23-Nov-99 Contains HIP, HSC70-INTERACTING PROTEIN(PROGESTERONE RECEPTOR-ASSOCIATED P48 PROTEIN), ESTs and STS. rxa01151958 GB_BA1: MTCY261 27322 Z97559 Mycobacterium tuberculosis H37Rvcomplete genome; segment Mycobacterium 38,789 17-Jun-98 95/162.tuberculosis GB_HTG4: AC009849 114993 AC009849 Drosophila melanogasterchromosome 2 clone BACR07H08 (D864) Drosophila melanogaster 39,21325-OCT-1999 RPCI-98 07.H.8 map 31B-31C strain y; cn bw sp, ***SEQUENCING IN PROGRESS ***, 55 unordered pieces. GB_HTG4: AC009849114993 AC009849 Drosophila melanogaster chromosome 2 clone BACR07H08(D864) Drosophila melanogaster 39,213 25-OCT-1999 RPCI-98 07.H.8 map31B-31C strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 55 unorderedpieces. rxa01161 1260 GB_BA2: AF176799 2943 AF176799 Lactobacilluspentosus PepQ (pepQ) and catabolite control protein A Lactobacilluspentosus 37,043 5-Sep-99 (ccpA) genes, complete cds. GB_BA2: AF0120843082 AF012084 Lactobacillus helveticus prolidase (pepQ) gene, completecds. Lactobacillus helveticus 46,796 1-Jul-98 GB_EST32: AI728955 611AI728955 BNLGHi12114 Six-day Cotton fiber Gossypium hirsutum cDNA 5′Gossypium hirsutum 37,647 11-Jun-99 similar to (AC004481) putativepermease [Arabidopsis thaliana], mRNA sequence. rxa01181 980 GB_BA1:MLCB22 40281 Z98741 Mycobacterium leprae cosmid B22. Mycobacteriumleprae 61,570 22-Aug-97 GB_BA1: MTCY190 34150 Z70283 Mycobacteriumtuberculosis H37Rv complete genome; segment Mycobacterium 60,43417-Jun-98 98/162. tuberculosis GB_BA1: SC5F7 40024 AL096872 Streptomycescoelicolor cosmid 5F7. Streptomyces coelicolor 57,011 22-Jul-99 A3(2)rxa01182 516 GB_HTG1: CEY116A8_2 110000 Z98858 Caenorhabditis eleganschromosome IV clone Y116A8, *** Caenorhabditis elegans 34,843 26-Oct-99SEQUENCING IN PROGRESS ***, in unordered pieces. GB_HTG1: CEY116A8_2110000 Z98858 Caenorhabditis elegans chromosome IV clone Y116A8, ***Caenorhabditis elegans 34,843 26-Oct-99 SEQUENCING IN PROGRESS ***, inunordered pieces. GB_IN1: CEY116A8C 260341 AL117204 Caenorhabditiselegans cosmid Y116A8C, complete sequence. Caenorhabditis elegans 34,84319-Nov-99 rxa01189 732 GB_BA1: D90915 130001 D90915 Synechocystis sp.PCC6803 complete genome, 17/27, 2137259-2267259. Synechocystis sp.36,538 7-Feb-99 GB_BA1: D90915 130001 D90915 Synechocystis sp. PCC6803complete genome, 17/27, 2137259-2267259. Synechocystis sp. 34,5127-Feb-99 GB_HTG3: AC010515 41038 AC010515 Homo sapiens chromosome 19clone LLNL-R_249H9, *** Homo sapiens 33,564 15-Sep-99 SEQUENCING INPROGRESS ***, 31 unordered pieces. rxa01192 681 GB_OM: CFP180RRC 5425X87224 Canis familiaris mRNA for ribosome receptor, p180. Canisfamiliaris 41,229 22-Jan-99 GB_OM: CFP180RRC 5425 X87224 Canisfamiliaris mRNA for ribosome receptor, p180. Canis familiaris 38,18722-Jan-99 rxa01214 1614 GB_IN1: CEY47D3A 199814 AL117202 Caenorhabditiselegans cosmid Y47D3A, complete sequence. Caenorhabditis elegans 36,60419-Nov-99 GB_PR4: AC006039 176257 AC006039 Homo sapiens clone NH0319F03,complete sequence. Homo sapiens 34,984 05-MAY-1999 GB_PR4: AC006039176257 AC006039 Homo sapiens clone NH0319F03, complete sequence. Homosapiens 35,951 05-MAY-1999 rxa01224 1146 GB_EST22: AI070047 479 AI070047UI-R-C1-In-f-08-0-UI.s1 UI-R-C1 Rattus norvegicus cDNA clone UI-R-Rattus norvegicus 36,975 5-Jul-99 C1-In-f-08-0-UI 3′, mRNA sequence.GB_RO: S75965 625 S75965 THP = Tamm-Horsfall protein {promoter} [rats,Genomic, 625 nt]. Rattus sp. 34,400 27-Jul-95 GB_EST5: H96951 459 H96951yu01g03.r1 Soares_pineal_gland_N3HPG Homo sapiens cDNA clone Homosapiens 32,969 11-DEC-1995 IMAGE: 232564 5′, mRNA sequence. rxa01250 588GB_PL1: NEULCCB 2656 M18334 N. crassa (strain TS) laccase gene, completecds. Neurospora crassa 44,330 03-MAY-1994 GB_OV: MTRACOMPL 16714 Y16884Rhea americana complete mitochondrial genome. Mitochondrion Rhea 35,09419-Jul-99 americana GB_OV: AF090339 16704 AF090339 Rhea americanamitochondrion, complete genome. Mitochondrion Rhea 35,094 27-MAY-1999americana rxa01277 2127 GB_PL2: AF111709 52684 AF111709 Oryza sativasubsp. indica Retrosat 1 retrotransposon and Ty3-Gypsy Oryza sativasubsp. indica 37,410 26-Apr-99 type Retrosat 2 retrotransposon, completesequences; and unknown genes. GB_IN1: CELZC250 34372 AF003383Caenorhabditis elegans cosmid ZC250. Caenorhabditis elegans 35,50614-MAY-1997 GB_EST1: Z14808 331 Z14808 CEL5E4 Chris Martin sorted cDNAlibrary Caenorhabditis elegans Caenorhabditis elegans 36,890 19-Jun-97cDNA clone cm5e4 5′, mRNA sequence. rxa01302 576 GB_BA1: MTCI65 34331Z95584 Mycobacterium tuberculosis H37Rv complete genome; segmentMycobacterium 59,298 17-Jun-98 50/162. tuberculosis GB_BA1: MSGY34840056 AD000020 Mycobacterium tuberculosis sequence from clone y348.Mycobacterium 59,227 10-DEC-1996 tuberculosis GB_BA1: SC5C7 41906AL031515 Streptomyces coelicolor cosmid 5C7. Streptomyces coelicolor39,261 7-Sep-98 rxa01303 1458 GB_BA1: TTAJ5043 837 AJ225043 Thermusthermophilus partial narK gene. Thermus thermophilus 55,245 18-Jun-98GB_PL2: AC010675 84723 AC010675 Arabidopsis thaliana chromosome I BACT17F3 genomic sequence, Arabidopsis thaliana 37,058 11-Nov-99 completesequence. GB_GSS9: AQ170862 518 AQ170862 HS_3165_B2_F03_T7 CIT ApprovedHuman Genomic Sperm Library Homo sapiens 38,610 17-OCT-1998 D Homosapiens genomic clone Plate = 3165 Col = 6 Row = L, genomic surveysequence. rxa01308 2503 GB_BA1: D90757 17621 D90757 Escherichia coligenomic DNA. (27.3-27.7 min). Escherichia coli 55,445 7-Feb-99 GB_BA1:D90787 15942 D90787 E. coli genomic DNA, Kohara clone #276(33.0-33.3min.). Escherichia coli 36,815 29-MAY-1997 GB_BA1: D90758 13860 D90758Escherichia coli genomic DNA. (27.6-27.9 min). Escherichia coli 54,9427-Feb-99 rxa01309 824 GB_BA1: SCJ12 35302 AL109989 Streptomycescoelicolor cosmid J12. Streptomyces coelicolor 62,423 24-Aug-99 A3(2)GB_BA1: BSNARYWI 12450 Z49884 B. subtilis nar[G, H, I, J, K], ywi[C, D,E] and argS genes. Bacillus subtilis 57,447 24-Jun-98 GB_BA1: BSUB0020212150 Z99123 Bacillus subtilis complete genome (section 20 of 21): from3798401 to Bacillus subtilis 37,129 26-Nov-97 4010550. rxa01358 1644GB_GSS11: AQ260413 453 AQ260413 CITBI-E1-2510B12.TF CITBI-E1 Homosapiens genomic clone Homo sapiens 41,531 24-OCT-1998 2510B12, genomicsurvey sequence. GB_EST20: AA840582 326 AA840582 vw77h07.r1 Stratagenemouse heart (#937316) Mus musculus cDNA Mus musculus 42,901 27-Feb-98clone IMAGE: 1261021 5′ similar to gb: J04181 Mouse A-X actin mRNA,complete cds (MOUSE);, mRNA sequence. GB_PAT: A39944 3836 A39944Sequence 1 from Patent WO9421807. unidentified 38,764 05-MAR-1997rxa01385 2004 GB_BA1: FVBPENTA 2519 M98557 Flavobacterium sp.pentachlorophenol 4-monooxygenase gene, Flavobacterium sp. 40,85526-Apr-93 complete mRNA. GB_PAT: I19994 2516 I19994 Sequence 2 frompatent U.S. Pat. No. 5512478. Unknown. 40,855 07-OCT-1996 GB_BA2:AF059680 2410 AF059680 Sphingomonas sp. UG30 pentachlorophenol4-monooxygenase Sphingomonas sp. UG30 42,993 27-Apr-99 (pcpB) gene,complete cds; and pentachlorophenol 4-monooxygenase reductase (pcpD)gene, partial cds. rxa01412 327 GB_GSS12: AQ332469 459 AQ332469HS_5003_A1_H08_SP6E RPCI11 Human Male BAC Library Homo Homo sapiens38,208 06-MAR-1999 sapiens genomic clone Plate = 579 Col = 15 Row = O,genomic survey sequence. GB_EST27: AA998532 453 AA998532UI-R-C0-ic-d-11-0-UI.s1 UI-R-C0 Rattus norvegicus cDNA clone UI-R-Rattus norvegicus 39,336 09-MAR-1999 C0-ic-d-11-0-UI 3′, mRNA sequence.GB_HTG1: HSA342D11 178183 AL121748 Homo sapiens chromosome 10 cloneRP11-342D11, *** Homo sapiens 40,550 23-Nov-99 SEQUENCING IN PROGRESS***, in unordered pieces. rxa01458 1173 GB_BA2: AE000745 15085 AE000745Aquifex aeolicus section 77 of 109 of the complete genome. Aquifexaeolicus 37,694 25-MAR-1998 GB_BA2: AE000745 15085 AE000745 Aquifexaeolicus section 77 of 109 of the complete genome. Aquifex aeolicus35,567 25-MAR-1998 rxa01571 723 GB_BA1: AB011413 12070 AB011413Streptomyces griseus genes for Orf2, Orf3, Orf4, Orf5, AfsA, Orf8,Streptomyces griseus 57,500 7-Aug-98 partial and complete cds. GB_BA1:AB011413 12070 AB011413 Streptomyces griseus genes for Orf2, Orf3, Orf4,Orf5, AfsA, Orf8, Streptomyces griseus 35,655 7-Aug-98 partial andcomplete cds. rxa01607 753 GB_PR4: AC005005 133893 AC005005 Homo sapiensPAC clone DJ412A9 from 22, complete sequence. Homo sapiens 38,39902-MAR-1999 GB_HTG3: AC008257 109187 AC008257 Drosophila melanogasterchromosome 2 clone BACR08A11 (D916) Drosophila melanogaster 33,74108-OCT-1999 RPCI-98 08.A.11 map 42A-42A strain y; cn bw sp, ***SEQUENCING IN PROGRESS ***, 93 unordered pieces. GB_HTG3: AC008257109187 AC008257 Drosophila melanogaster chromosome 2 clone BACR08A11(D916) Drosophila melanogaster 33,741 08-OCT-1999 RPCI-98 08.A.11 map42A-42A strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 93 unorderedpieces. rxa01609 996 GB_BA1: MTV003 13246 AL008883 Mycobacteriumtuberculosis H37Rv complete genome; segment Mycobacterium 39,36917-Jun-98 125/162. tuberculosis GB_BA1: MSGB1529CS 36985 L78824Mycobacterium leprae cosmid B1529 DNA sequence. Mycobacterium leprae60,624 15-Jun-96 GB_BA1: AB024601 14807 AB024601 Pseudomonas aeruginosadapD gene for tetrahydrodipicolinate N- Pseudomonas aeruginosa 41,60312-MAR-1999 succinyletransferase, complete cds, strain PAO1. rxa016541119 GB_GSS4: AQ704352 532 AQ704352 HS_2147_A2_H04_MR CIT Approved HumanGenomic Sperm Homo sapiens 37,838 7-Jul-99 Library D Homo sapiensgenomic clone Plate = 2147 Col = 8 Row = O, genomic survey sequence.GB_RO: MMAE000663 250611 AE000663 Mus musculus TCR beta locus from bases1 to 250611 (section 1 of Mus musculus 35,799 4-Sep-97 3) of thecomplete sequence. GB_EST23: AI158428 511 AI158428 ud24f12.r1 Soares2NbMT Mus musculus cDNA clone Mus musculus 41,337 30-Sep-98 IMAGE:1446863 5′, mRNA sequence. rxa01664 945 GB_OV: AF026198 63155 AF026198Fugu rubripes neural cell adhesion molecule L1 homolog (L1-CAM) Fugurubripes 35,187 02-MAY-1998 gene, complete cds; putative protein 1(PUT1) gene, partial cds; mitosis-specific chromosome segregationprotein SMC1 homolog (SMC1) gene, complete cds; and calcium channelalpha-1 subunit homolog (CCA1) and putative protein 2 (PUT2) genes,partial cds, complete sequence. GB_PR3: AC004466 122186 AC004466 Homosapiens 12q13.1 PAC RPCI5-1057I20 (Roswell Park Cancer Homo sapiens37,382 17-Sep-98 Institute Human PAC library) complete sequence. GB_PR3:AC004466 122186 AC004466 Homo sapiens 12q13.1 PAC RPCI5-1057I20 (RoswellPark Cancer Homo sapiens 37,325 17-Sep-98 Institute Human PAC library)complete sequence. rxa01795 720 GB_BA2: CGU13922 4412 U13922Corynebacterium glutamicum putative type II 5-cytosoine Corynebacterium99,444 3-Feb-98 methyltransferase (cgIIM) and putative type IIrestriction endonuclease glutamicum (cgIIR) and putative type I or typeIII restriction endonuclease (clgIIR) genes, complete cds. GB_BA1:S86113 1044 S86113 ORF 1 [Neisseria gonorrhoeae, Genomic, 1044 nt].Neisseria gonorrhoeae 58,320 07-MAY-1993 GB_PAT: I22080 850 I22080Sequence 1 from patent U.S. Pat. No. 5525717. Unknown. 57,72207-OCT-1996 rxa01802 954 GB_BA2: AE001519 14062 AE001519 Helicobacterpylori, strain J99 section 80 of 132 of the complete Helicobacter pyloriJ99 33,510 20-Jan-99 genome. GB_GSS5: AQ774071 552 AQ774071HS_2269_B1_C10_T7C CIT Approved Human Genomic Sperm Homo sapiens 37,96729-Jul-99 Library D Homo sapiens genomic clone Plate = 2269 Col = 19 Row= F, genomic survey sequence. GB_PR4: AC007459 40907 AC007459 Homosapiens chromosome 16 clone 306C6, complete sequence. Homo sapiens39,140 04-MAY-1999 rxa01838 842 GB_BA1: SCE15 26440 AL049707Streptomyces coelicolor cosmid E15. Streptomyces coelicolor 36,29722-Apr-99 GB_HTG3: AC009545 165042 AC009545 Homo sapiens chromosome 11clone 131_J_04 map 11, *** Homo sapiens 37,651 01-OCT-1999 SEQUENCING INPROGRESS ***, 8 unordered pieces. GB_HTG3: AC009545 165042 AC009545 Homosapiens chromosome 11 clone 131_J_04 map 11, *** Homo sapiens 37,65101-OCT-1999 SEQUENCING IN PROGRESS ***, 8 unordered pieces. rxa01848 867GB_BA1: MTCY24A1 20270 Z95207 Mycobacterium tuberculosis H37Rv completegenome; segment Mycobacterium 38,270 17-Jun-98 124/162. tuberculosisGB_EST21: C89252 587 C89252 C89252 Mouse early blastocyst cDNA Musmusculus cDNA clone Mus musculus 37,219 28-MAY-1998 01B00061JC08, mRNAsequence. GB_EST14: AA423340 457 AA423340 ve39d04.r1 Soares mousemammary gland NbMMG Mus musculus Mus musculus 38,377 16-OCT-1997 cDNAclone IMAGE: 820519 5′, mRNA sequence. rxa01849 1224 GB_BA1: MTCY24A120270 Z95207 Mycobacterium tuberculosis H37Rv complete genome; segmentMycobacterium 39,950 17-Jun-98 124/162. tuberculosis GB_BA2: RCPHSYNG45959 Z11165 R. capsulatus complete photosynthesis gene cluster.Rhodobacter capsulatus 37,344 2-Sep-99 GB_BA1: RSP010302 40707 AJ010302Rhodobacter sphaeroides photosynthetic gene cluster. Rhodobactersphaeroides 40,898 27-Aug-99 rxa01868 2049 GB_BA1: MTV033 21620 AL021928Mycobacterium tuberculosis H37Rv complete genome; segment Mycobacterium38,679 17-Jun-98 11/162. tuberculosis GB_BA1: MLCL622 42498 Z95398Mycobacterium leprae cosmid L622. Mycobacterium leprae 38,911 24-Jun-97GB_BA1: MSGB983CS 36788 L78828 Mycobacterium leprae cosmid B983 DNAsequence. Mycobacterium leprae 38,933 15-Jun-96 rxa01885 924 GB_BA1:MTCY1A10 25949 Z95387 Mycobacterium tuberculosis H37Rv complete genome;segment Mycobacterium 51,094 17-Jun-98 117/162. tuberculosis GB_PR3:HSU220B11 41247 Z69908 Human DNA sequence from cosmid cU220B11, betweenmarkers Homo sapiens 39,038 23-Nov-99 DXS6791 and DXS8038 on chromosomeX. GB_BA1: PDU17435 993 U17435 Paracoccus denitrificans Fnr-liketranscriptional activator (nnr) gene, Paracoccus denitrificans 39,39019-Jul-95 complete cds. rxa01914 526 GB_PR3: AC005796 43843 AC005796Homo sapiens chromosome 19, cosmid R31408, complete sequence. Homosapiens 34,961 06-OCT-1998 GB_PR3: HS390C10 114231 AL008721 Homo sapiensDNA sequence from BAC 390C10 on chromosome Homo sapiens 39,600 23-Nov-9922q11.21-12.1. Contains an Immunoglobulin LIKE gene and a pseudogenesimilar to Beta Crystallin. Contains ESTs, STSs, GSSs and taga and tatrepeat polymorphisms, complete sequence. GB_PR3: AC005796 43843 AC005796Homo sapiens chromosome 19, cosmid R31408, complete sequence. Homosapiens 37,725 06-OCT-1998 rxa01932 1020 GB_PR3: AC003025 112309AC003025 Human Chromosome 11p12.2 PAC clone pDJ466a11, complete Homosapiens 35,585 23-Jul-98 sequence. GB_GSS3: B78728 312 B78728CIT-HSP-431E3.TV CIT-HSP Homo sapiens genomic clone 431E3, Homo sapiens38,907 25-Jun-98 genomic survey sequence. GB_PR3: AC003025 112309AC003025 Human Chromosome 11p12.2 PAC clone pDJ466a11, complete Homosapiens 35,859 23-Jul-98 sequence. rxa01933 726 GB_HTG1: HS74O16 169401AL110119 Homo sapiens chromosome 21 clone RPCIP704O1674 map 21q21, Homosapiens 35,302 27-Aug-99 *** SEQUENCING IN PROGRESS ***, in unorderedpieces. GB_HTG1: HS74O16 169401 AL110119 Homo sapiens chromosome 21clone RPCIP704O1674 map 21q21, Homo sapiens 35,302 27-Aug-99 ***SEQUENCING IN PROGRESS ***, in unordered pieces. GB_PR4: AC006032 170282AC006032 Homo sapiens BAC clone NH0115E20 from Y, complete sequence.Homo sapiens 37,640 27-Feb-99 rxa01971 954 GB_HTG3: AC008230 108469AC008230 Drosophila melanogaster chromosome 2 clone BACR17I17 (D934)Drosophila melanogaster 35,466 10-Aug-99 RPCI-98 17.I.17 map 53A-53Cstrain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 108 unorderedpieces. GB_HTG3: AC008230 108469 AC008230 Drosophila melanogasterchromosome 2 clone BACR17I17 (D934) Drosophila melanogaster 35,46610-Aug-99 RPCI-98 17.I.17 map 53A-53C strain y; cn bw sp, *** SEQUENCINGIN PROGRESS***, 108 unordered pieces. GB_PR3: AF064860 165382 AF064860Homo sapiens chromosome 21q22.3 PAC 70I24, complete sequence. Homosapiens 39,716 2-Jun-98 rxa02016 900 GB_EST2: D48846 459 D48846RICS15292A Rice green shoot Oryza sativa cDNA, mRNA sequence. Oryzasativa 37,118 2-Aug-95 GB_GSS10: AQ195886 595 AQ195886 RPCI11-66O13.TJRPCI-11 Homo sapiens genomic clone RPCI-11- Homo sapiens 41,00020-Apr-99 66O13, genomic survey sequence. GB_GSS10: AQ195886 595AQ195886 RPCI11-66O13.TJ RPCI-11 Homo sapiens genomic clone RPCI-11-Homo sapiens 34,790 20-Apr-99 66O13, genomic survey sequence. rxa02017807 GB_EST20: AA855266 406 AA855266 vw70b08.r1 Stratagene mouse heart(#937316) Mus musculus cDNA Mus musculus 42,638 06-MAR-1998 clone IMAGE:1260279 5′, mRNA sequence. GB_EST20: AA855266 406 AA855266 vw70b08.r1Stratagene mouse heart (#937316) Mus musculus cDNA Mus musculus 37,18306-MAR-1998 clone IMAGE: 1260279 5′, mRNA sequence. rxa02018 1073GB_BA1: SC5C7 41906 AL031515 Streptomyces coelicolor cosmid 5C7.Streptomyces coelicolor 41,732 7-Sep-98 GB_BA1: MTCI65 34331 Z95584Mycobacterium tuberculosis H37Rv complete genome; segment Mycobacterium62,395 17-Jun-98 50/162. tuberculosis GB_BA1: SCJ12 35302 AL109989Streptomyces coelicolor cosmid J12. Streptomyces coelicolor 61,60324-Aug-99 A3(2) rxa02048 1497 GB_PAT: E15823 2323 E15823 DNA encodingcell surface protein from Corynebacterium Corynebacterium 53,94228-Jul-99 ammoniagenes. ammoniagenes GB_OM: SSAMPTDN 3387 Z29522 S.scrofa mRNA for aminopeptidase N. Sus scrofa 42,672 26-Sep-94 GB_OV:D87992 3181 D87992 Gallus gallus mRNA for aminopeptidase Ey, completecds. Gallus gallus 41,554 5-Jun-99 rxa02101 1386 GB_BA1: AP000064 247695AP000064 Aeropyrum pernix genomic DNA, section 7/7. Aeropyrum pernix39,882 22-Jun-99 GB_PL2: ATAC006587 79262 AC006587 Arabidopsis thalianachromosome II BAC T17D12 genomic sequence, Arabidopsis thaliana 38,49023-MAR-1999 complete sequence. GB_PL2: ATAC006587 79262 AC006587Arabidopsis thaliana chromosome II BAC T17D12 genomic sequence,Arabidopsis thaliana 34,863 23-MAR-1999 complete sequence. rxa02265 423GB_BA2: AF120718 4137 AF120718 Lactobacillus fermentum urease operon,partial sequence. Lactobacillus fermentum 56,265 31-MAR-1999 GB_PAT:E03531 2896 E03531 DNA sequence coding for acid urease. Lactobacillusfermentum 56,265 29-Sep-97 GB_BA1: LBAAURE 2896 D10605 L. fermentum genefor acid urease. Lactobacillus fermentum 56,265 2-Feb-99 rxa02276 801GB_GSS10: AQ242920 451 AQ242920 HS_2061_A1_E08_MR CIT Approved HumanGenomic Sperm Homo sapiens 37,916 03-OCT-1998 Library D Homo sapiensgenomic clone Plate = 2061 Col = 15 Row = I, genomic survey sequence.GB_IN1: SLMMTPMF 14503 D29637 Physarum polycephalum mitochondrial DNA.Mitochondrion Physarum 40,335 12-MAY-1999 polycephalum GB_IN2: AF0122495542 AF012249 Physarum polycephalum strain aux2-S region of mitochondriaderived Mitochondrion Physarum 40,335 08-MAY-1998 from mF plasmid,including URFA′, URFC, URFD, URFE, URFF, and polycephalum URFG genes,complete cds, and URFH gene, partial cds. rxa02277 738 GB_BA2: AF048784681 AF048784 Actinomyces naeslundii urease accessory protein (ureG)gene, Actinomyces naeslundii 66,814 9-Feb-99 complete cds. GB_BA2:AF056321 5482 AF056321 Actinomyces naeslundii urease gamma subunit UreA(ureA), urease Actinomyces naeslundii 63,686 9-Feb-99 beta subunit UreB(ureB), urease alpha subunit UreC (ureC), urease accessory protein UreE(ureE), urease accessory protein UreF (ureF), urease accessory proteinUreG (ureG), and urease accessory protein UreD (ureD) genes, completecds. GB_BA2: SSU35248 5773 U35248 Streptococcus salivarius ure clusternickel transporter homolog (urel) Streptococcus salivarius 61,93126-Jan-96 gene, partial cds, and urease beta subunit (ureA), gammasubunit (ureB), alpha subunit (ureC), and accessory proteins (ureE),(ureF), (ureG), and (ureD) genes, complete cds. rxa02278 972 GB_GSS3:B49054 543 B49054 RPCI11-4I13.TV RPCI-11 Homo sapiens genomic cloneRPCI-11- Homo sapiens 39,161 8-Apr-99 4I13, genomic survey sequence.GB_PL1: PMCMSGI 3363 L27092 Pneumocystis carinii B-cell receptor (msgl)gene, 3′ end. Pneumocystis carinii 39,819 26-Sep-94 GB_PL2: AF03855612792 AF038556 Pneumocystis carinii f. sp. hominis variant regions ofmajor surface Pneumocystis carinii f. sp. 33,832 10-Sep-98 glycoproteins(msg1, msg3, msg4) genes, partial cds. hominis rxa02317 735 GB_GSS8:AQ051031 914 AQ051031 nbxb0004dG10r CUGI Rice BAC Library Oryza sativagenomic clone Oryza sativa 32,299 24-MAR-1999 nbxb0004N20r, genomicsurvey sequence. GB_GSS8: AQ051031 914 AQ051031 nbxb0004dG10r CUGI RiceBAC Library Oryza sativa genomic clone Oryza sativa 34,573 24-MAR-1999nbxb0004N20r, genomic survey sequence. rxa02334 746 GB_BA1: CGU350233195 U35023 Corynebacterium glutamicum thiosulfate sulfurtransferase(thtR) gene, Corynebacterium 100,000 16-Jan-97 partial cds, acyl CoAcarboxylase (accBC) gene, complete cds. glutamicum GB_BA1: MTCY71 42729Z92771 Mycobacterium tuberculosis H37Rv complete genome; segmentMycobacterium 60,380 10-Feb-99 141/162. tuberculosis GB_BA1: U0001233312 U00012 Mycobacterium leprae cosmid B1308. Mycobacterium leprae37,660 30-Jan-96 rxa02351 1039 GB_HTG2: HS225E12 126464 AL031772 Homosapiens chromosome 6 clone RP1-225E12 map q24, *** Homo sapiens 35,97303-DEC-1999 SEQUENCING IN PROGRESS ***, in unordered pieces. GB_HTG2:HS225E12 126464 AL031772 Homo sapiens chromosome 6 clone RP1-225E12 mapq24, *** Homo sapiens 35,973 03-DEC-1999 SEQUENCING IN PROGRESS ***, inunordered pieces. GB_HTG2: HS225E12 126464 AL031772 Homo sapienschromosome 6 clone RP1-225E12 map q24, *** Homo sapiens 36,99203-DEC-1999 SEQUENCING IN PROGRESS ***, in unordered pieces. rxa02410789 GB_BA1: AB020624 1605 AB020624 Corynebacterium glutamicum murl genefor D-glutamate racemase, Corynebacterium 99,227 24-Jul-99 complete cds.glutamicum GB_EST4: H51527 294 H51527 yo33b09.s1 Soares adult brainN2b4HB55Y Homo sapiens cDNA Homo sapiens 40,411 18-Sep-95 clone IMAGE:179705 3′, mRNA sequence. GB_GSS1: CNS003CM 1101 AL064136 Drosophilamelanogaster genome survey sequence T7 end of BAC # Drosophilamelanogaster 37,674 3-Jun-99 BACR08C19 of RPCI-98 library fromDrosophila melanogaster (fruit fly), genomic survey sequence. rxa02477744 GB_HTG4: AC010054 130191 AC010054 Drosophila melanogaster chromosome3L/74E2 clone RPCI98-15E10, Drosophila melanogaster 37,466 16-OCT-1999*** SEQUENCING IN PROGRESS ***, 70 unordered pieces. GB_HTG4: AC010054130191 AC010054 Drosophila melanogaster chromosome 3L/74E2 cloneRPCI98-15E10, Drosophila melanogaster 37,466 16-OCT-1999 *** SEQUENCINGIN PROGRESS ***, 70 unordered pieces. GB_HTG4: AC009375 137069 AC009375Drosophila melanogaster chromosome 3L/75A1 clone RPCI98-44L18,Drosophila melanogaster 39,118 16-OCT-1999 *** SEQUENCING IN PROGRESS***, 59 unordered pieces. rxa02513 832 GB_BA1: MTER260 373 X92572 M.terrae gene for 32 kDa protein (partial). Mycobacterium terrae 42,89515-Jan-98 GB_PL1: AB019229 84294 AB019229 Arabidopsis thaliana genomicDNA, chromosome 3, P1 clone: Arabidopsis thaliana 36,084 20-Nov-99MDC16, complete sequence. GB_PL1: AB019229 84294 AB019229 Arabidopsisthaliana genomic DNA, chromosome 3, P1 clone: Arabidopsis thaliana35,244 20-Nov-99 MDC16, complete sequence. rxa02531 834 GB_BA1: CGLATTB271 X89850 C. glutamicum DNA for attB region. Corynebacterium 40,5908-Aug-96 glutamicum GB_EST11: AA239557 423 AA239557 mv25f04.r1GuayWoodford Beier mouse kidney day 0 Mus musculus Mus musculus 38,76012-MAR-1997 cDNA clone IMAGE: 656095 5′ similar to gb: X52634 Murine tlmoncogene for tlm protein (MOUSE);, mRNA sequence. GB_BA1: RSPYPPCL 6500AJ002398 Rhodobacter sphaeroides pyp and pcl genes, and orfA, orfB,orfC, Rhodobacter sphaeroides 37,091 17-DEC-1998 orfD, orfE, orfF.rxa02548 314 GB_BA2: AF127374 63734 AF127374 Streptomyces lavendulaeLinA homolog, cytochrome P450 Streptomyces lavendulae 66,242 27-MAY-1999hydroxylase ORF4, cytochrome P450 hydroxylase ORF3, MitT (mitT), MitS(mitS), MitR (mitR), MitQ (mitQ), MitP (mitP), MitO (mitO), MitN (mitN),MitM (mitM), MitL (mitL), MitK (mitK), MitJ (mitJ), MitI (mitI), MitH(mitH), MitG (mitG), MitF (mitF), MitE (mitE), MitD (mitD), MitC (mitC),MitB (mitB), MitA (mitA), MmcA (mmcA), MmcB (mmcB), MmcC (mmcC), MmcD(mmcD), MmcE (mmcE), MmcF (mmcF), MmcG (mmcG), MmcH (mmcH), MmcI (mmcI),MmcJ (mmcJ), MmcK (mmcK), MmcL (mmcL), MmcM (mmcM), MmcN (mmcN), MmcO(mmcO), Mrd (mrd), MmcP (mmcP), MmcQ (mmcQ), MmcR (mmcR), MmcS (mmcS),MmcT (mmcT), MmcU (mmcU), MmcV (mmcV), Mct (mct), MmcW (mmcW), MmcX(mmcX), and MmcY (mmcY) genes, complete cds; and unknown genes. GB_BA2:AF127374 63734 AF127374 Streptomyces lavendulae LinA homolog, cytochromeP450 Streptomyces lavendulae 38,411 27-MAY-1999 hydroxylase ORF4,cytochrome P450 hydroxylase ORF3, MitT (mitT), MitS (mitS), MitR (mitR),MitQ (mitQ), MitP (mitP), MitO (mitO), MitN (mitN), MitM (mitM), MitL(mitL), MitK (mitK), MitJ (mitJ), MitI (mitI), MitH (mitH), MitG (mitG),MitF (mitF), MitE (mitE), MitD (mitD), MitC (mitC), MitB (mitB), MitA(mitA), MmcA (mmcA), MmcB (mmcB), MmcC (mmcC), MmcD (mmcD), MmcE (mmcE),MmcF (mmcF), MmcG (mmcG), MmcH (mmcH), MmcI (mmcI), MmcJ (mmcJ), MmcK(mmcK), MmcL (mmcL), MmcM (mmcM), MmcN (mmcN), MmcO (mmcO), Mrd (mrd),MmcP (mmcP), MmcQ (mmcQ), MmcR (mmcR), MmcS (mmcS), MmcT (mmcT), MmcU(mmcU), MmcV (mmcV), Mct (mct), MmcW (mmcW), MmcX (mmcX), and MmcY(mmcY) genes, complete cds; and unknown genes. GB_GSS4: AQ741886 742AQ741886 HS_5569_B2_B02_SP6 RPCI-11 Human Male BAC Library Homo Homosapiens 38,907 16-Jul-99 sapiens genomic clone Plate = 1145 Col = 4 Row= D, genomic survey sequence. rxa02558 1098 GB_EST18: AA567307 741AA567307 HL01004.5prime HL Drosophila melanogaster head BlueScriptDrosophila melanogaster 38,736 28-Nov-98 Drosophila melanogaster cDNAclone HL01004 5prime, mRNA sequence. GB_EST27: AI402394 630 AI402394GH21610.5prime GH Drosophila melanogaster head pOT2 DrosophilaDrosophila melanogaster 41,308 8-Feb-99 melanogaster cDNA clone GH216105prime, mRNA sequence. GB_GSS10: AQ237646 715 AQ237646 RPCI11-61I9.TJBRPCI-11 Homo sapiens genomic clone RPCI-11- Homo sapiens 44,34021-Apr-99 61I9, genomic survey sequence. rxa02565 1389 GB_EST32:AI726448 562 AI726448 BNLGHi5854 Six-day Cotton fiber Gossypium hirsutumcDNA 5′ Gossypium hirsutum 37,003 11-Jun-99 similar to (U53418)UDP-glucose dehydrogenase [Glycine max], mRNA sequence. GB_EST32:AI726198 608 AI726198 BNLGHi5243 Six-day Cotton fiber Gossypium hirsutumcDNA 5′ Gossypium hirsutum 40,925 11-Jun-99 similar to (U53418)UDP-glucose dehydrogenase [Glycine max], mRNA sequence. GB_PR4: AC002992154848 AC002992 Homo sapiens chromosome Y, clone 203M13, completesequence. Homo sapiens 38,039 13-OCT-1999 rxa02574 1131 GB_EST4: H29653415 H29653 ym58f01.r1 Soares infant brain 1NIB Homo sapiens cDNA cloneHomo sapiens 39,036 17-Jul-95 IMAGE: 52678 5′ similar to SP: OXDD_BOVINP31228 D-ASPARTATE OXIDASE;, mRNA sequence. GB_PR3: HSDJ261K5 131974AL050350 Human DNA sequence from clone 261K5 on chromosome 6q21-22.1.Homo sapiens 35,957 23-Nov-99 Contains the 3′ part of the gene for anovel organic cation transporter (BAC ORF RG331P03), the DDO gene forD-aspartate oxidase (EC 1.4.3.1), ESTs, STSs, GSSs and two putative CpGislands, complete sequence. GB_EST2: R20147 494 R20147 yg18h02.r1 Soaresinfant brain 1NIB Homo sapiens cDNA clone Homo sapiens 36,437 17-Apr-95IMAGE: 32866 5′ similar to SP: OXDD_BOVIN P31228 D-ASPARTATE OXIDASE;,mRNA sequence. rxa02589 888 GB_HTG1: CEY6E2 186306 Z96799 Caenorhabditiselegans chromosome V clone Y6E2, *** Caenorhabditis elegans 37,97902-OCT-1997 SEQUENCING IN PROGRESS ***, in unordered pieces. GB_HTG1:CEY6E2 186306 Z96799 Caenorhabditis elegans chromosome V clone Y6E2, ***Caenorhabditis elegans 37,979 02-OCT-1997 SEQUENCING IN PROGRESS ***, inunordered pieces. GB_HTG3: AC011690 72277 AC011690 Homo sapiens clone17_E_13, LOW-PASS SEQUENCE SAMPLING. Homo sapiens 35,814 10-OCT-1999rxa02592 894 GB_BA1: MSGB983CS 36788 L78828 Mycobacterium leprae cosmidB983 DNA sequence. Mycobacterium leprae 53,235 15-Jun-96 GB_GSS9:AQ170723 487 AQ170723 HS_2270_B2_F05_MR CIT Approved Human Genomic SpermLibrary Homo sapiens 39,666 16-OCT-1998 D Homo sapiens genomic clonePlate = 2270 Col = 10 Row = L, genomic survey sequence. GB_GSS12:AQ349397 791 AQ349397 RPCI11-118H16.TJ RPCI-11 Homo sapiens genomicclone RPCI-11- Homo sapiens 34,204 07-MAY-1999 118H16, genomic surveysequence. rxa02603 1119 GB_BA1: MTV026 23740 AL022076 Mycobacteriumtuberculosis H37Rv complete genome; segment Mycobacterium 37,97524-Jun-99 157/162. tuberculosis GB_IN2: AC005714 177740 AC005714Drosophila melanogaster, chromosome 2R, region 58D4-58E2, BAC Drosophilamelanogaster 41,226 01-MAY-1999 clone BACR48M13, complete sequence.GB_EST19: AA775050 218 AA775050 ac76e10.s1 Stratagene lung (#937210)Homo sapiens cDNA clone Homo sapiens 40,826 5-Feb-98 IMAGE: 868554 3′similar to gb: Y00371_rna1 HEAT SHOCK COGNATE 71 KD PROTEIN (HUMAN);,mRNA sequence. rxa02630 1446 GB_BA1: MLCL373 37304 AL035500Mycobacterium leprae cosmid L373. Mycobacterium leprae 49,015 27-Aug-99GB_BA1: MTV044 16150 AL021999 Mycobacterium tuberculosis H37Rv completegenome; segment Mycobacterium 49,192 17-Jun-98 45/162. tuberculosisGB_BA1: MLU15180 38675 U15180 Mycobacterium leprae cosmid B1756.Mycobacterium leprae 45,621 09-MAR-1995 rxa02643 1167 GB_EST37: AI950576308 AI950576 wx52e08.x1 NCI_CGAP_Lu28 Homo sapiens cDNA clone Homosapiens 40,909 6-Sep-99 IMAGE: 2547302 3′, mRNA sequence. GB_EST37:AI950576 308 AI950576 wx52e08.x1 NCI_CGAP_Lu28 Homo sapiens cDNA cloneHomo sapiens 40,288 6-Sep-99 IMAGE: 2547302 3′, mRNA sequence. rxa02644774 GB_EST34: AV149547 302 AV149547 AV149547 Mus musculus C57BL/6J 10-11day embryo Mus musculus Mus musculus 38,627 5-Jul-99 cDNA clone2810489D03, mRNA sequence. GB_EST35: AV156221 271 AV156221 AV156221 Musmusculus head C57BL/6J 12-day embryo Mus Mus musculus 33,990 7-Jul-99musculus cDNA clone 3000001C24, mRNA sequence. GB_EST32: AV054919 274AV054919 AV054919 Mus musculus pancreas C57BL/6J adult Mus musculus Musmusculus 36,585 23-Jun-99 cDNA clone 1810033C08, mRNA sequence. rxa02745902 GB_BA1: MTV007 32806 AL021184 Mycobacterium tuberculosis H37Rvcomplete genome; segment Mycobacterium 39,298 17-Jun-98 64/162.tuberculosis GB_BA2: AF027770 30683 AF027770 Mycobacterium smegmatisFxbA (fxbA) gene, partial cds; FxbB (fxbB), Mycobacterium smegmatis55,125 03-DEC-1998 FxbC (fxbC), and FxuD (fxtD) genes, complete cds; andunknown genes. GB_BA2: SAU43537 3938 U43537 Streptomyces argillaceusmithramycin resistance determinant, ATP- Streptomyces argillaceus 46,8685-Sep-96 binding protein (mtrA) and membrane protein (mtrB) genes,complete cds. rxa02746 290 GB_BA1: CAJ10319 5368 AJ010319Corynebacterium glutamicum amtP, glnB, glnD genes and partial ftsYCorynebacterium 100,000 14-MAY-1999 and srp genes. glutamicum GB_BA1:MTCY338 29372 Z74697 Mycobacterium tuberculosis H37Rv complete genome;segment Mycobacterium 39,785 17-Jun-98 127/162. tuberculosis GB_HTG3:AC008733 216140 AC008733 Homo sapiens chromosome 19 cloneCITB-E1_2525J15, *** Homo sapiens 35,688 3-Aug-99 SEQUENCING IN PROGRESS***, 72 unordered pieces. rxa02820 1411 GB_BA1: BFU64514 3837 U64514Bacillus firmus dppABC operon, dipeptide transporter protein dppABacillus firmus 36,859 1-Feb-97 gene, partial cds, and dipeptidetransporter proteins dppB and dppC genes, complete cds. GB_IN1: CET04C1020958 Z69885 Caenorhabditis elegans cosmid T04C10, complete sequence.Caenorhabditis elegans 35,934 2-Sep-99 GB_EST35: AI823090 720 AI823090L30-944T3 Ice plant Lambda Uni-Zap XR expression library, 30 hoursMesembryanthemum 35,770 21-Jul-99 NaCl treatment Mesembryanthemumcrystallinum cDNA clone L30- crystallinum 944 5′ similar to 60Sribosomal protein L36 (AC004684)[Arabidopsis thaliana], mRNA sequence.rxa02834 518 GB_BA1: CJY13333 3315 Y13333 Campylobacter jejuni clpBgene. Campylobacter jejuni 53,400 12-Apr-99 GB_BA2: AF065404 181654AF065404 Bacillus anthracis virulence plasmid PX01, complete sequence.Bacillus anthracis 45,168 20-OCT-1999 GB_PL2: AC006601 110684 AC006601Arabidopsis thaliana chromosome V map near 60.5 cM, complete Arabidopsisthaliana 36,680 22-Feb-99 sequence.

1. An isolated polypeptide selected from the group consisting of: a) anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:2;b) an isolated polypeptide comprising a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:2; c) an isolated polypeptide which is encoded by a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1; d) anisolated polypeptide which is encoded by a nucleic acid moleculecomprising a nucleotide sequence which is at least 50% identical to theentire nucleotide sequence of SEQ ID NO:1; e) an isolated polypeptidecomprising an amino acid sequence which is at least 50% identical to theentire amino acid sequence of SEQ ID NO:2; and f) an isolatedpolypeptide comprising a fragment of a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, wherein said polypeptide fragmentmaintains a biological activity of the polypeptide comprising the aminosequence.
 2. The isolated polypeptide of claim 1, further comprisingheterologous amino acid sequences.
 3. The isolated polypeptide of claim1, wherein the polypeptide has a sulfate adenylate transferase subunit 2activity.
 4. A method for producing a fine chemical, comprisingculturing a recombinant host cell capable of expressing the polypeptideof claim
 1. 5. The method of claim 4, wherein said method furthercomprises the step of recovering the fine chemical from said culture. 6.The method of claim 4, wherein said cell belongs to the genusCorynebacterium or Brevibacterium.
 7. The method of claim 4, whereinsaid cell is selected from the group consisting of Corynebacteriumglutamicum, Corynebacterium herculis, Corynebacterium, lilium,Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum,Corynebacterium acetophilum, Corynebacterium ammoniagenes,Corynebacterium fujiokense, Corynebacterium nitrilophilus,Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibacteriumdivaricatum, Brevibacterium flavum, Brevibacterium healii,Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum,Brevibacterium lactofermentum, Brevibacterium linens, Brevibacteriumparaffinolyticum, and those strains set forth in Table
 3. 8. The methodof claim 4, wherein expression of the polypeptide results in modulationof production of said fine chemical.
 9. The method of claim 4, whereinsaid fine chemical is selected from the group consisting of organicacids, proteinogenic and nonproteinogenic amino acids, purine andpyrimidine bases, nucleosides, nucleotides, lipids, saturated andunsaturated fatty acids, diols, carbohydrates, aromatic compounds,vitamins, cofactors, polyketides, and enzymes.
 10. The method of claim4, wherein said fine chemical is an amino acid.
 11. The method of claim10, wherein the amino acid is selected from the group consisting oflysine, glutamate, glutamine, alanine, aspartate, glycine, serine,threonine, methionine, cysteine, valine, leucine, isoleucine, arginine,proline, histidine, tyrosine, phenylalanine, and tryptophan.
 12. Amethod for diagnosing the presence or activity of Corynebacteriumdiphtheriae in a subject, comprising detecting the presence of at leastone of the polypeptide molecules of claim 1 thereby diagnosing thepresence or activity of Corynebacterium diphtheriae in the subject.